Soluble lymphotoxin beta receptor and anti-lymphotoxin receptor and ligand antibodies as therapeutic agents for treatment

ABSTRACT

Compositions and methods comprising “lymphotoxin-β receptor blocking agents” which block lymphotoxin-β receptor signaling and are useful for altering immunological diseases, and particularly antibody mediated immune responses.

TECHNICAL FIELD OF THE INVENTION

[0001] This invention relates to compositions and methods comprising“lymphotoxin-β receptor blocking agents” which block lymphotoxin-βreceptor signaling. Lymphotoxin-β receptor blocking agents are usefulfor treating immunological diseases, more specifically for inhibitingantibody mediated immune responses, regulating the expression ofaddressing and cell trafficking, and influencing the differentiation offollicular dendritic cells. This invention relates to soluble forms ofthe lymphotoxin-β receptor extracellular domain, and antibodies directedagainst either the lymphotoxin-β receptor or its ligand, surfacelymphotoxin, that act as lymphotoxin-β receptor blocking agents.

BACKGROUND OF THE INVENTION

[0002] There are two arms of acquired immunity, which, while able tocollaborate to achieve the common goal of eliminating antigen, aremediated by distinct participants of the immune system with differenteffects. One arm of acquired immune response, humoral immunity, ismediated primarily by B cells and circulating antibodies. The other arm,referred to as cellular or cell-mediated immunity, is mediated by Tcells that synthesize and elaborate cytokines which affect other cells.

[0003] Activation and differentiation of B cells in response to mostantigens requires that (1) B cells receive an antigen signal via theirantigen specific receptor, membrane Ig, and (2) B cells receive contactdependent and independent signals from activated T cells. The contactdependent costimulatory signal results from ligation of the CD40receptor on B cells to the CD40 ligand expressed on activated T helpercells. (Laman et al., Crit. Rev. Immunol., 16, pp. 59-108 (1996); VanKooten and Banchereau, Adv. Immunol., 61, pp. 1-77 (1996)). Contactindependent signaling is mediated by cytokines synthesized andelaborated by activated T cells. Together these contact dependent andindependent signals drive B cells to differentiate to either (1) memoryB cells poised to mediate a more rapid response upon secondary exposureto antigen, or (2) antibody secreting plasma cells. Plasma cells, whichare the terminal differentiation stage of B cells, synthesize andsecrete antibodies.

[0004] T helper cells (“Th”) play several significant roles in theimmune system. Cytokines elaborated by Th cells at the onset of animmune challenge have been shown to affect which immune effectorpathways are subsequently activated. Th cells are activated by theinteraction of their antigen specific receptor with antigen-presentingcells (APCs) displaying on their surfaces peptide fragments of processedforeign antigen in association with MHC class II molecules. Activated Thcells, in turn, secrete cytokines (lymphokines) which activate theappropriate immune effector mechanisms.

[0005] Th cells can be divided into three subgroups ThO, Th1 and Th2,based upon their cytokine secretion patterns. (Fitch et al., Ann. Rev.Immunol., 11, pp. 29-48 (1993)). In mice, non-stimulated “naive” Thelper cells produce IL-2. Short term stimulation of Th cells leads toThO precursor cells, which produce a wide range of cytokines includingIFN-α, IL-2, IL-4, IL-5 and IL-10. Chronically-stimulated ThO cells candifferentiate into either Th1 or Th2 cell types, whereupon the cytokineexpression pattern changes. Certain cytokines, for example IL-3, GM-CSFand TNF, are released by both Th1 and Th2 cells. Other cytokines aremade exclusively by only one Th cell subgroup. (Romagnani et al., Ann.Rev. Immunol., 12, pp. 227-57 (1994)). Th1 cells produce LTα IL-2 andIFN-γ which activate macrophages and inflammatory responses associatedwith cellular immunity and resistance to intracellular infections.

[0006] Th2 cells produce the cytokines IL-4, IL-5, IL-6 and IL-10 whichincrease eosinophil and mast cell production and promote the fullexpansion and maturation of B cells. (Howard et al., “T cell-derivedcytokines and their receptors”, Fundamental Immunology, 3d ed., RavenPress, New York (1993)). Th2 cells also participate in generating B cellmemory, somatic mutation and thus affinity maturation, and in regulatingde novo immunoglobulin isotype switching. For example, the Th2 cytokineIL-4 switches activated B cells to the IgG1 isotype while suppressingother isotypes. IL-4 also stimulates the overproduction of IgE in type Ihypersensitivity reactions. The Th2 cytokine IL-5 induces the IgAisotype important in mucosal immunity.

[0007] The secondary lymphoid tissues, such as the lymph nodes (LN),spleen and mucosal lymphoid tissues, are highly efficient in trappingand concentrating foreign substances, and are the main sites of antigendriven activation and differentiation of T and B lymphocytes. Theseprocesses are dependent upon the diversity and organization of cells inthese tissues, providing a framework for many aspects of humoral immuneresponses, such as T/B cell interactions, germinal center (GC)formation, affinity maturation, immunoglobulin class switching and celltrafficking. (Klein, J., Immunology, John Wiley and sons, (1982)). Themolecular mechanisms responsible for the development, structuralmaintenance and function of peripheral lymphoid tissues are not fullyunderstood.

[0008] Although the general structure of the secondary lymphoid tissuesdiffers markedly and shows variations between species of mammalia, thefine structure of these secondary lymphoid tissues shares certainfeatures, such as, for example: (1) antigen accessibility, (2)structural features ensuring continued contact of antigen withlymphocytes, (3) T cell rich areas surrounded by B cells, (4) B cellrich follicles, (5) marginal zone type sites, (6) specializedendothelial cells, and (7) antibody production sites, as discussed infurther detail below.

[0009] The secondary lymphoid tissues are accessible to antigen in thesystem. For example, antigen accesses the spleen via the sinusoidalblood supply, the LN via the afferent lymphatic vessels, and istransported across specialized epithelium into the mucosal lymphoidtissue.

[0010] The secondary lymphoid tissues in various species also sharecertain structural features such as follicular dendritic cells (FDC) andinterdigitating cells (IDC), which ensure the continued presence ofantigen in the lymphocyte rich areas of the tissues.

[0011] Another common feature is the presence of T cell rich areassurrounded by B cells. T cell rich areas include, for example, theperiarteriolar lymphoid sheaths in the white pulp of the spleen, and theparacortical region of LN, which contain large numbers of recirculatingT cells and IDC, which in turn function as accessory cells for T and Bcells.

[0012] Additionally, lymphoid tissues typically have B cell rich primaryand secondary follicles in the white pulp of the spleen, and in thecortex of the LN. Secondary follicles in such lymphoid tissues are alsocalled germinal centers (GC) and have a dense FDC network to capture andpresent antigens.

[0013] Marginal—zone type areas are also noted as defined histologicareas in the murine spleen and more diffuse sites in human secondarylymphoid organs. These areas are comprised primarily of marginal zonemacrophages (MZM), metallophilic macrophages (MM), marginal zone B cellsand reticular cells, but may also include T cells and dendritic cells.(Kraal, Int. Rev. Cytol. 132, pp.31-74 (1992)). The opening of thearterial blood stream into the marginal zone areas gives antigens directaccess to these cells and promotes cellular reactions to antigens atthis site. (Kraal, Int. Rev. Cytol. 132, pp.31-74 (1992)). The presenceof MZM are also required for optimal trafficking of B cells in thesplenic white pulp. (Kraal, 1992; Kraal, et al, Immunology, 68,pp.227-232(1989)).

[0014] Typically, blood lymphocytes enter the secondary lymphoid tissuesby crossing specialized endothelium, for example the endothelial liningof the venules of LN (high endothelial venules -HEV) and the endotheliallining of splenic blood sinusoids in the marginal zone—like structures.This endothelium expresses adhesion molecules and addressins whichfunction in the trafficking of cells to secondary lymphoid tissues. Forexample, peripheral LN addressins (PNAd) are distinct from the mucosalLN addressin, MAdCAM-1, which is involved in trafficking of lymphocytesto mucosal lymphoid tissues, including tissues such as the mesentericLN, Peyer's patches and lamina propria.

[0015] Not all addressins are clearly defined, for example, theaddressin for lymphocyte homing to spleen remains undefined. Thephysiological roles of these addressins include enhancing recruitment ofappropriate sets of antigen specific lymphocytes into an immuneresponse, and subsequent dissemination of the immune response throughoutthe body.

[0016] Finally, the plasma cells, which are the antibody producingplasma cells, are detected at different locations from where theprogenitor B cells are activated by antigen. For example, antibodyproduced by plasma cells in splenic red pulp mainly results from B cellactivation in T cell zones, and plasma cells in the medulla of LN arederived from B cells activated in T cell zones of the same node.Similarly, antibody produced by plasma cells in bone marrow arederivatives of B cells activated in spleen and lymph node, and plasmacells in the lamina propria of gut mainly derive from B cells activatedin mesenteric LN or gut associated lymphoid tissue.

[0017] See e.g., ICM MacLennan, “The Structure and Function of SecondaryLymphoid Tissues” in Clinical Aspects of Immunology 5th edition, eds. P.J. Lachman, Sir D. K. Peters, F. S. Rosen, M. J. Walport, BlackwellScientific Publications pp 13-30 (1993).

[0018] In general, the cellular/histologic events underlying a humoralimmune response to T dependent antigens are as follows (Toellner, etal., J. Exp. Med., 183, pp. 2303-2312 (1996)):

[0019] In the Inductive phase, naive B and T cells are activated andrecruited into the immune response in the days immediately after antigenenters the body. In the spleen, for example, within 12 hours ofimmunization for a secondary response, memory B cells encounterblood-borne antigen in the marginal zone and leave the marginal zone togo to the T cell zones. B cells can be detected in the T cell zoneswithin 24 hours. Immunoglobulin switch transcripts can be detectedwithin 12 hours of secondary antigen exposure, thus indicating that theT-B cell interaction has already occurred. The B cells then migrate tothe exit zones and red pulp where they proliferate to form foci of Bcell blasts and differentiate into plasma cells. The B cells alsocontinue to proliferate in the IDC rich T cell zone. Within 4 days afterimmunization, and after proliferation in the GC, B memory cellproduction will start. In a primary response, well developed GC areapparent by day 10 and reach peak size by day 14 post-immunization Tcell proliferation in the T cell zones becomes evident 48-72 hours andpeaks on day 7 after immunization. This T cell proliferation contributesto T cell dependent B cell activation. Proliferative levels in the Tcell zone decrease as GC forms. T cell proliferation also occurs in theGC where centrocytes (B cells) in the dark zone pick up antigen fromIDC, and present antigen to T cells in light zone.

[0020] T cell dependent antigen can activate marginal zone B cells,newly produced naive B cells and recirculating lymphocytes attracted toand retained in secondary lymphoid organs by addressins and adhesionmolecules. Naive B cells show the same kinetics for going to T cellzone, etc. as do the activated B cells.

[0021] Established Phase of T Cell Dependent Responses

[0022] The established phase of T cell dependent responses is maintainedby the continued activation of memory B cells in the follicles ofsecondary lymphoid organs. There is very little recruiting of naive Bcells at this stage, and the response is primarily driven by antigenretained on FDC. GC are required for optimal memory generation, isotypeswitching, somatic mutation and thus affinity maturation ofimmunoglobulin.

[0023] The mounting of such lymphocyte responses results in theproduction of antibodies able to circulate throughout the body byvarious routes, for example, antibodies leave the spleen via the blood,and exit LN via the efferent lymphatics. The antibodies thus encounterand directly bind to the invading pathogen. This recognition event setsoff a cascade of immune effector mechanisms, including activation of thecomplement cascade and cellular reactions to mediate protection of thehost from the pathogen.

[0024] Antibodies also play a role in some pathologic responses such ashypersensitivity responses—inappropriate or disproportionate immuneresponses evoked upon contact with a previously encountered antigen.There are four recognized types of hypersensitivity.

[0025] Type I “immediate hypersensitivity” involves allergen-induced Th2cell activation and Th2 cytokine release. The Th2 cytokine IL-4stimulates B cells to undergo isotype switching to produce IgE, which inturn activates mast cells to produce acute inflammatory reactions suchas those which lead to eczema, asthma and rhinitis.

[0026] Types II and III hypersensitivity are caused by IgG and IgMantibodies directed against cell surface antigens or specific tissueantigens (Type II) or soluble serum antigens to form circulating immunecomplexes (Type III).

[0027] Type IV “delayed type” hypersensitivity (DTH) is a Th1 cellmediated response and can be transferred between mice by transferringTh1 cells, but not by transferring serum alone. This featuredistinguishes Type IV DTH from the other three types ofhypersensitivity, which require humoral immune responses causedprimarily by antibodies which can be transferred in cell-free serum.(Roitt et al., Immunology, pp. 19.1-22.12 (Mosby-Year Book Europe Ltd.,3d ed. 1993))

[0028] Pathological humoral immune responses are associated with anumber of organ-specific and systemic autoimmune conditions such asSystemic Lupus Erythematosus, Wegener's Granulomatosis, PolyarteritisNodosa (PAN), Rapidly Progressive Crescentic Glomerulonephritis andIdiopathic Thrombocytopenia Purpura, as well as chronic inflammatorydiseases such as the Graves' and Chagas' disease. Humoral immuneresponses may also contribute to grafted tissue and transplanted organrejection.

[0029] The treatment of these various immunological conditions to datehas generally employed immunomodulatory and immunosuppressive agents.Three general immunosuppressive agents currently used are steroids,cyclophosphamide and azathioprine.

[0030] Steroids are pleiotropic anti-inflammatory agents which suppressactivated macrophages and inhibit the activity of antigen presentingcells in ways which reverse many pathologic T cell effects.Cyclophosphamide, an alkylating agent, mediates cell death by inhibitingDNA replication and repair. Azathioprine is an anti-proliferative agentwhich inhibits DNA synthesis. These non-specific immunosuppressiveagents are generally required in high doses which increase theirtoxicity (e.g. nephro- and hepatotoxicity) and cause adverse sideeffects. They are thus unsuitable for long term therapies.

[0031] Thus, there is an unmet need for additional agents and therapieswhich overcome the problems caused by conventional treatments.

SUMMARY OF THE INVENTION

[0032] The present invention solves the problems referred to above byproviding pharmaceutical compositions and methods for treatingimmunological diseases by inhibiting lymphotoxin-β receptor (LT-β-R)signaling using lymphotoxin-β receptor blocking agents. Moreparticularly, the compositions and methods comprising LT-β-R blockingagents are useful for inhibiting antibody mediated immune responses, forregulating addressin expression levels and cell trafficking, forinfluencing the differentiation of follicular dendritic cells, and foraltering the structural organization of secondary lymphoid tissues andsimilar lymphoid structures arising in pathologic conditions such as,for example, systemic lupus erythematosis and idiopathicthrombocytopenia purpura. Additionally, in certain embodiments theclaimed invention is useful for altering the association between immunecomplexes and B cells. More specifically, the methods of the inventioncan prevent the presentation or deposition of antigens on cells, oralternatively, to essentially dissolve or erase the antigens alreadypresent on cells.

[0033] In alternative embodiments, the LT-β-R blocking agent is selectedfrom the group consisting of soluble lymphotoxin-β-R, an antibodydirected against LT-β-R, and an antibody directed agains surface LTligand.

[0034] In one embodiment, soluble forms of the lymphotoxin-β receptorextracellular domain that act as LT-β-R blocking agents are provided.The preferred compositions and methods of this embodiment comprise arecombinant lymphotoxin-β receptor fusion protein that has the LT-β-Rextracellular ligand binding domain fused to an immunoglobulin constantheavy chain domain. More preferably, the LT-β-R ligand binding domain isfused to a human IgG Fc domain.

[0035] In another embodiment of this invention, antibodies that act asLT-β-R blocking agents are provided. Preferred compositions and methodsof this embodiment comprise one or more antibodies directed against thelymphotoxin-β receptor. More preferably, the antibody is a monoclonalantibody. Other preferred compositions and methods of this embodimentcomprise one or more antibodies directed against surface lymphotoxin.More preferably, the antibody is a monoclonal antibody directed againstlymphotoxin-β. Preferred antibodies include the anti-human BT-β-R mAbBDA8, and anti-human LT-β mAb B9.

[0036] In yet other emboidments, the claimed invention relates tomethods of altering the humoral immune response in an animal byadministering a pharmaceutical composition which has a therapeuticallyeffective amount of a LT-β-R blocking agent. In certain otherembodiments, the pharmaceutical composition is administered in an amountsufficient to coat LT-β-R positive cells for about 1 to about 14 days.The pharmaceutical composition may in certain embodiments furthercomprise a pharmaceutically acceptable carrier or adjuvant.

[0037] In other embodiments, the claimed methods inhibit LT-β-Rsignaling without inhibiting TNF-R signaling, using the LT-β-R blockingagents described above. Methods of treating, preventing, or eliminatingthe human immunodeficiency virus in a mammal are also encompasses in theclaimed invention, comprising administration of blocking agents ofLT-β-R either alone, or in conjunction with pharmaceutical carriers,adjuvants, or other drugs known to those skilled in the art to be usefulin the treatment or amelioration of the symptoms of HIV or AIDS.

[0038] Additionally, the present invention relates to methods oftreatment in the transplantation field, i.e.graft rejection.Specifically, certain embodiments relate to the coadministration of ablocking agent of the CD40 pathway and a blocking agent of the LTpathway.

BRIEF DESCRIPTION OF THE DRAWINGS

[0039]FIG. 1 is a sequence of the extracellular portion of the human LTβreceptor which encodes the ligand binding domain.

[0040]FIG. 2 is an immunohistochemical analysis of the spleen of micewhich received multiple injections of LTβ-R-Ig or LFA-3-Ig fusionproteins, and antigen.

[0041]FIG. 3 is an immunohistochemical analysis showing the absence ofgerminal centers from spleens of LTβ-R-Ig treated and MR-1 (anti-CD40ligand antibody) treated mice, and the presence of follicular dendriticcells in spleens of MR-1 but not LTβ-R-Ig treated mice. Fusion proteinsand SRBC antigen were administered as described for FIG. 2.

[0042]FIG. 4 is an immunohistochemical analysis showing addressinexpression is altered in LN of mice treated in utero and continuouslypost-birth with LTβ-R-Ig.

[0043]FIG. 5 is an immunohistochemical analysis of Lymphocytepositioning and expression of macrophage markers in mesenteric LN ofmice treated (as for FIG. 4) in utero and continuously post-birth withLTβ-R-Ig

[0044]FIG. 6 is an immunohistochemical analysis showing that LTβ-R-Igtreatment of mice inhibits the antibody response to SRBC.

[0045]FIG. 7 is a representation of immune complex trapping on FDCs.

DETAILED DESCRIPTION OF THE INVENTION

[0046] In order that the invention herein described may be fullyunderstood, the following detailed description is set forth.

[0047] The terms “immunoglobulin response” or “humoral response” as usedherein refer to the immunological response of an animal to a foreignantigen whereby the animal produces antibodies to the foreign antigen.The Th2 class of T helper cells are important to the efficientproduction of high affinity antibodies.

[0048] The term “germinal center” as used herein refers to a secondary Bcell follicle which forms after antigen immunization. The appearance ofthis histologic site correlates with optimal memory generation, isotypeswitching, somatic hypermutation and thus the affinity maturation of anantibody response.

[0049] The terms “marginal zone” or “marginal—zone type area” refer tohistologically described compartments of the secondary lymphoid tissuescomprised primarily of marginal zone macrophages (MZM), metallophilicmacrophages (MM), marginal zone B cells and reticular cells, and also Tcells and dendritic cells. The arterial blood stream opens into themarginal sinuses thus giving antigens direct access to these cells andpromoting cellular reactions to antigens at this site.

[0050] The term “addressin” as used herein refers to a molecule involvedin the homing of lymphocytes to secondary lymphoid organs. Suchmolecules are expressed on endothelial cells, specifically the highendothelial venules in the lymph nodes. The splenic addressin isundefined. MAdCAM-1 is a mucosal addressin; PNAd is a peripheraladdressin.

[0051] The term “T helper (Th) cells” as used herein, refers to afunctional subclass of T cells which help to generate cytotoxic T cellsand which cooperate with B cells to stimulate antibody production.Helper T cells recognize antigen in association with class II MHCmolecules and provide contact dependent and contact independent(cytokine) signals to effector cells.

[0052] The term “cytokine”, as used herein, refers to a molecule whichmediates signaling between cells. A “lymphokine” is a cytokine releasedby lymphocytes.

[0053] The term “Th2” refers to a subclass of T helper cells thatproduce LTα, interferon-γ and IL-2 (and other cytokines) and whichelicit inflammatory reactions associated with a cellular, i.e.non-immunoglobulin, response to a challenge.

[0054] The term “Th2” refers to a subclass of T helper cells thatproduces cytokines, such as IL-4, IL-5, IL-6 and IL-10, which areassociated with an immunoglobulin (humoral) response to an immunechallenge.

[0055] The term “Fc domain” of an antibody refers to a part of themolecule comprising the hinge, CH2 and CH3 domains, but lacking theantigen binding sites. The term is also meant to include the equivalentregions of an IgM or other antibody isotype.

[0056] The term “anti-LTβ receptor antibody” refers to any antibody thatspecifically binds to at least one epitope of the LTβ receptor.

[0057] The term “anti-LT antibody” refers to any antibody thatspecifically binds to at least one epitope of LTα, LTβ or a LTα/βcomplex.

[0058] The term “LTβ-R signaling” refers to molecular reactionsassociated with the LTβ-R pathway and subsequent molecular reactionswhich result therefrom.

[0059] The term “ILTβ-R blocking agent” refers to an agent that candiminish ligand binding to LTβ-R, cell surface LTβ-R clustering or LTβ-Rsignaling, or that can influence how the LTβ-R signal is interpretedwithin the cell.

[0060] A LTβ-R blocking agent that acts at the step of ligand-receptorbinding can inhibit LT ligand binding to the LTβ-R by at least 20%.Examples of LTβ-R blocking agents include soluble LTβ-R-Fc molecules,and anti-LT α, anti-LTβ, anti-LT α/β and anti-LTβ-R Abs. Preferably, theantibodies do not cross-react with the secreted form of LT α.

[0061] The term “LTβ-R biological activity” refers to: 1) the ability ofthe LTβ-R molecule or derivative to compete for soluble or surface LTligand binding with soluble or surface LTβ-R molecules; or 2)native LTβactivity such as the ability to stimulate an immune regulatory responseor cytotoxic activity.

[0062] The term “LT ligand” refers to a LT α/β heteromeric complex orderivative thereof that can specifically bind to the LTβ receptor.

[0063] The term “ILTβ-R ligand binding domain” refers to the portion orportions of the LTβ-R that are involved in specific recognition of andinteraction with a LT ligand.

[0064] The terms “surface LT” and “surface LT complex” refer to acomplex comprising LT a and membrane-bound LTβ subunits—includingmutant, altered and chimeric forms of one or more of the subunits—whichis displayed on the cell surface.

[0065] “Surface LT ligand” refers to a surface LT complex or derivativethereof that can specifically bind to the LTβ receptor.

[0066] The term “subject” refers to an animal, or to one or more cellsderived from an animal. Preferably, the animal is a mammal. Cells may bein any form, including but not limited to cells retained in tissue, cellclusters, immortalized, transfected or transformed cells, and cellsderived from an animal that has been physically or phenotypicallyaltered.

[0067] Lymphotoxin β: A Member of the TNF Family

[0068] Tumor Necrosis Factor (TNF)-related cytokines have emerged as alarge family of pleiotropic mediators of host defense and immuneregulation. Members of this family exist in membrane-bound forms whichact locally through cell-cell contact, or as secreted proteins which canact on distant targets. A parallel family of TNF-related receptorsreacts with these cytokines and triggers a variety of pathways includingcell death, cell proliferation, tissue differentiation andproinflammatory responses.

[0069] TNF, lymphotoxin a (LTα, also called TNFB) and lymphotoxin β(LTB) are members of the TNF family of ligands, which also includes theligands to the Fas, CD27, CD30, CD40, OX-40 and 4-1BB receptors. (Smithet al., Cell, 76, pp. 959-62 (1994)). Signaling by several members ofthe TNF family—including TNF, LTα, LTβ and Fas—can induce tumor celldeath by necrosis or apoptosis (programmed cell death). Innon-tumorigenic cells, TNF and many of the TNF family ligand-receptorinteractions influence immune system development and responses tovarious immune challenges.

[0070] Most membrane-associated LTα/β complexes (“surface LT”) have aLTα1/β2 stoichiometry. (Browning et al., Cell, 72, pp. 847-56 (1993);Browning et al., J. Immunol., 154, pp. 33-46 (1995)). Surface LT ligandsdo not bind TNF-R with high affinity and do not activate TNF-Rsignaling. The LTβ receptor (LTβ-R), does however bind these surfacelymphotoxin complexes with high affinity (Crowe et al., Science, 264,pp. 707-10 (1994)).

[0071] LTβ-R signaling, like TNF-R signaling, has anti-proliferativeeffects and can be cytotoxic to tumor cells. In applicants' co-pendingU.S. application Ser. No. 08/378,968, compositions and methods forselectively stimulating LTβ-R using LTβ-R activating agents aredisclosed. LTβ-R activating agents are useful for inhibiting tumor cellgrowth without co-activating TNF-R-induced proinflammatory orimmunoregulatory pathways.

[0072] Recent gene targeting studies suggest a role for LTα/β in thedevelopment of secondary lymphoid organs. (Banks et al., J. Immunol.,155, pp. 1685-1693 (1995); De Togni et al., Science, 264, pp. 703-706(1994)). Indeed, LTa-deficient mice lack lymph nodes (LN) and Peyer'spatches (PP). Moreover, their spleens have disrupted architecture andthe expression of functional markers on cells of the splenic marginalzone is altered. (Banks et al., 1995; De Togni et al., Science, 264, pp.703-706 (1994), Matsumoto et al., Science, 271, pp. 1289-1291 (1996)).None of these characteristics have been described for either of the TNFreceptor knock out mice. (Erickson et al., Nature, 372, pp.560-563(1994); Pfeffer et al., Cell, 73, pp. 457-467 (1993); Rothe et al.,Nature, 364, pp. 798-802 (1993). Applicants have recently defined a rolefor membrane LTα/β complexes in secondary lymphoid organ development byshowing that the progeny of mice which had been injected duringgestation with a soluble form of mouse LTP-R fused to the human IgG1 Fcportion (LTβ-R-Ig) lacked most lymph nodes and showed disrupted splenicarchitecture. (Rennert et al, 1996, “Surface Lymphotoxin alpha/betacomplex is required for the development of peripheral lymphoid organs.”J. Exp Med, 184: 1999-2006). In another study, mice transgenic for asimilar LTβ-R-Ig construct which starts to be expressed three days afterbirth, were shown to have LN. However, their splenic architecture wasdisrupted and several markers of splenic marginal zone cells were notexpressed (Ettinger et al., “Disrupted splenic architecture, but normallymph node development in mice expressing a soluble LTβ-R/IgG1 fusionprotein”., Proc. Natl. Acac. Sci. U.S.A. 93: 13102-7). Together thesedata indicate there is a temporal requirement for membrane LT functionsto mediate effects on the development of secondary lymphoid organs, butnot for effects on splenic architecture.

[0073] The TNF system may also function in development of the spleen.Splenic marginal zone cells of TNF-deficient mice do not expressmacrophage markers or MAdCAM-1 (Alexopoulou et al., 60th Int. TNFCongress, Eur. Cytokine Network, pp. 228 (1996); Pasparakis et al., 60thInt. TNF Congress, Eur. Cytokine Network, pp. 239 (1996)).TNF-R55-deficient mice also lack MAdCAM-1 (but not MOMA-1) staining inthe splenic marginal zone. (Neumann et al., J. Exp. Med., 184, pp.259-264 (1996), Matsumoto et al., Science, 271, pp. 1289-1291 (1996)).The expression of these markers as seen in the spleen ofTNF-R75-deficient mice appears normal. (Matsumoto et al., Science, 271,pp. 1289-1291 (1996)).

[0074] Lymphoid-like tissues do not only arise as a part ofdevelopmental processes but also appear under some pathologicalcircumstances such as chronic inflammation, a process recently termedneolymphoorganogenesis. (Picker and Butcher, Annu. Rev. Immunol., 10,pp. 561-591 (1992), Kratz, et al., J. Exp. Med., 183, pp. 1461-1471(1996)). Such processes are apparently influenced by TNF family members.Mice transgenic for the LTα gene driven by the rat insulin promoter(RIP-LT) developed LT-induced chronic inflammatory lesions withcharacteristics of organized lymphoid tissues. (Kratz, et al., J. Exp.Med., 1183, pp. 1461-1471 (1996); Picarella et al., Proc. Natl. Acad.Sci., 89, pp. 10036-10040 (1992)).

[0075] The evaluation of LT function during a T cell—dependent immuneresponse, using LTα-deficient mice, showed the necessity of LT for GCformation, possibly for maintaining an organized follicular dendriticcell (FDCs) structure, and for humoral responses. (Banks et al., J.Immunol., 155, pp. 1685-1693 (1995); Matsumoto et al., Science, 271, pp.1289-1291 (1996); Matsumoto et al., Nature, 382, pp. 462-466 (1996)).TNF-R55-deficient mice also lack FDCs, fail to develop GC and fail todevelop an optimal antibody response to sheep red blood cells (SRBC).This suggests that TNF-R55 might be triggered by soluble LT or TNFsignals for most of these responses (Le Hir et al., J. Exp. Med., 183,pp. 2367-2372 (1996), Alexopoulou et al., 60th Int. TNF Congress, Eur.Cytokine Network, pp. 228 (1996); Pasparakis et al., 60th Int. TNFCongress, Eur. Cytokine Network, pp. 239 (1996)). A functional role forthe surface LT/LTβ-R pathway in the humoral immune responses has to datebeen undefined.

[0076] The LTβ receptor, a member of the TNF family of receptors,specifically binds to surface LT ligands. LTβ-R binds LT heteromericcomplexes (predominantly LTα1/β2 and LTα2/β1) but does not bind TNF orLTα (Crowe et al., Science, 264, pp. 707-10 (1994)). LTβ-R mRNAs arefound in the human spleen, thymus and in general organs with immunesystem involvement. Although studies on LTβ-R expression are in theirearly stages, LTβ-R expression patterns appear to be similar to thosereported for TNF-R55 except that LTβ-R is lacking on peripheral blood Tand B cells and T and B cell lines.

[0077] Cell surface lymphotoxin (LT) complexes have been characterizedin CD4⁺ T cell hybridoma cells (II-23.D7) which express high levels ofLT. (Browning et al., J. Immunol., 147, pp. 1230-37 (1991); Androlewiczet al., J. Biol. Chem., 267, pp. 2542-47 (1992), both of which areherein incorporated by reference). The expression and biological rolesof LTβ-R, LT subunits and surface LT complexes have been reviewed by C.F. Ware et al. “The ligands and receptors of the lymphotoxin system”, inPathways for Cytolysis, Current Topics Microbiol. Immunol.,Springer-Verlag, pp. 175-218 (1995) specifically incorported byreference herein.

[0078] LTα expression is induced and LTα secreted primarily by activatedT and B lymphocytes and natural killer (NK) cells. Among the T helpercells, LTα appears to be produced by Th1 but not Th2 cells. LTα has alsobeen detected in melanocytes. Microglia and T cells in lesions ofmultiple sclerosis patients can also be stained with anti-LTα antisera(Selmaj et al., J. Clin. Invest., 87, pp. 949-954 (1991)).

[0079] Lymphotoxin β (also called p33) is expressed on the surface ofhuman and mouse T lymphocytes, T cell lines, B cell lines andlymphokine-activated killer (LAK) cells. LTβ is the subject ofapplicants' co-pending international applications PCT/US91/04588,published Jan. 9, 1992 as WO 92/00329; and PCT/US93/11669, publishedJun. 23, 1994 as WO 94/13808, which are herein incorporated byreference.

[0080] Surface LT complexes are primarily expressed by activated T(helper, Th1, and killer cells) and B lymphocytes and natural killer(NK) cells as defined by FACS analysis or immunohistology using anti-LTαantibodies or soluble LTβ-R-Ig fusion proteins. In applicants copendingU.S. application Ser. No. 08/505,606, filed Jul. 21, 1995, compositionsand methods for using soluble LTβ receptors and anti-LTβ receptor andligand specific antibodies as therapeutics for the treatment ofimmunological diseases mediated by Th1 cells are disclosed. Surface LThas also been described on human cytotoxic T lymphocyte (CTL) clones,activated peripheral mononuclear lymphocytes (PML), IL-2—activatedperipheral blood lymphocytes (LAK cells), pokeweed mitogen-activated oranti-CD40—activated peripheral B lymphocytes (PBL) and various lymphoidtumors of T and B cell lineage. Engagement of alloantigen-bearing targetcells specifically induces surface LT expression by CD8⁺ and CD4⁺ CTLclones.

[0081] Applicants have described herein several immunological functionsfor surface LT, and show the effects of LTα/β binding reagents on thegeneration and character of immunoglobulin responses, maintenance of thecellular organization of secondary lymphoid tissues including effects onthe differentiation state of follicular dendritic cells and germinalcenter formation, and addressin expression levels which influence celltrafficking. Thus applicants define therapeutic applications for surfaceLTα/β and LTp receptor binding agents.

[0082] Until the present invention, however, the impact of LT-β-Rsignaling on humoral, or immunogenic, responses has not been fullyunderstood. The inventors have, for the first time, discovered thatblocking the LT pathway, either LT-β or LT-β-R can alter the humoralimmune response in an animal. Thus, the claimed invention in a broadembodiment relates to methods of altering the humoral immune response inan animal comprising the steps of administering a pharmaceuticalcomposition which comprises a therapeutically effective amount of ablocking agent of the LT pathway, specifically preferred, LT-β-Rblockers.

[0083] Any blocking agent can be used in the invention, and one skilledin the art can easily determine agents which block the LT-β-R. Forexample, such blocking agents may include small molecule inhibitors ofthe receptor, soluble lymphotoxin-β-Receptor, antibodies directedagainst the LT-β-R, and antibodies directed against the surface LTligand. In preferred embodiments, the blocking agents comprise a solubleLT-β-R having a ligand binding domain that can selectively bind to asurface LT ligand, and, more preferably, where the soluble LT-β-Rcomprises a human immunoglobulin FC domain.

[0084] In other embodiments, preferred blocking agents includemonoclonal antibodies directed against the LT-β-R, including,preferably, anti-human LT-β-R mAb BDA8, and anit-human LT-β mAb B9. Morepreferred antibodies include A1.D5.18 and AO.D12.10. and BB-F6In certaininstances, it may be desirable to use a monclonal antibody directedagainst a murine surface LT ligand.

[0085] The long term presentation of antigen by FDCs is likely to beimportant in autoimmune diseases where the continual activation of theimmune system by endogenous or autoantigens perpetuates the disease.Immune complex trapping on FDC's is illustrated in FIG. 7. The abilityto remove these immune complexes from the FDC would serve to reduce theamount of immune activation and dampen the disease or even stop diseaseprogression. Those autoiimune diseases that involve abberant antibodyresponses are obvious targets for LT pathway inhibitors although othermore “classically” T cell mediated autoimmune diseases may haveunrecognized humoral components, and therefore may also be beneficiallyimpacted.

[0086] Likewise, in the transplantation field, graft rejection, i.e.,host vs. graft disease and graft vs. host disease, require thepresentation of antigen to perpetuate. The mechanisms described here formanipulating FDC may also apply to those problems associated with therecognition of non-self, i.e. transplantation.

[0087] Additionally, the continued presentation of antigen ormaintenance of antigen memory may play a role in those autoimmunediseases caused by molecular mimicry. For example, the immune reactionto the lyme disease infectious agent Borrelia burgdorferi leads to anarthritis-like disease presumably because saome antigenic epitope onthis bacterium resembles a normal joint component. Removal of theFDC-retained lyme bacterium antigen may ameliorate lyme disease inducedarthritis. Such therapy would also be relevant to other cases of mimicryassociated with infectious agents.

[0088] Applicants have surprisingly found that the administration ofblocking agents of LT-β-R are capable of interfering with thepresentation and/or deposition of antigens on follicular dendriticcells. Typically, B cells recognize antigen as immune complexes bound tothe surface of follicular dendritic cells. Follicular dendritic cellsmay retain the antigens for an unspecified period of time. Periodicalcontact with the antigen retained on the FDC thus may be related to thememory retention of B cells. Thus, the claimed methods encompassnumerous disease states which are dependent upon the presentation ofantigen on dendritic cells. The administration of blocking agents of theinvention can be done prior to introduction of antigen into an animal,in which case the blocking agents will prevent all or a portion of thedeposition of the antigen on the follicular dendritic cells, therebypreventing, or diminishing, the expected immunogenic response.Alternatively, the blocking agents of the invention can be administeredto an animal at a point after the follicular dendritic cells haveantigen associated with them. Applicants claimed methods can disruptthis association, such that the expected immunogenic response would thenbe diminished or abolished. Thus, the therapeutic methods of theinvention can involve the elimination in whole or in part of the immunecomplexes already trapped in B cell follicles, or, the prevention, inwhole or in part, of the trapping of immune complexes on B cellfollicles.

[0089] The ability to disrupt the association between these antigenpresenting follicular dendritic cells and the immune complexes appearsto be unique to the LT-β pathway. For example, anti-CD40L (MR-1) isanother member of the TNF family and is also expressed on folliculardendritic cells. Like LT-β-R/Ig, MR-1 has been shown to prevent germinalcell formation, however, does not affect the expression of FDC markers.Anti-CD40-L, unlike LT-β-R, does not prevent immune complex trapping onfollicular dendritic cells, nor is it able to eliminate immune complexespreviously trapped on follicular dendritic cells. Additionally,applicants have shown that anti-CD40-L does not affect thesurvival/maintenance of previously generated memory B cells.

[0090] Although the precise basis for the differences beteween theimpace of anti-CD-40L and LT-β-R blocking agents is not known, it ishypothesized that CD40 may provide survival signals to B cells. However,the LT system is critical to maintain follicular dendritic cells in afully differentiated and functional state, a condition which appears tobe necesary for germinal center reaction and memory B cell generationand maintenance. Thus, blocking the CD4G/CD40L pathway may preventgeneration of memory B cells, but will not affect the alreadyestablished memory B cell pool. Blocking the LT pathway, on the otherhand, prevents not only the generation and maintenance of memory Bcells, but also affects the maintenance of previously generated memory Bcells.

[0091] A further appliction of inhibition of the LT pathway lies in thetreatment of viruses that form reservoirs in the follicular dendriticcell (FDC) compartment. The HIV virus is a good example of such a case.Following viral infection, large amounts of infectious virus reside onFDC's in the B cell follicles of the secondary lymphoid organs. (Heatheet al., 1995, “Follicular dendritic cells and human immunodeficiencyvirus infectivity.”, Nature 377: 740-4). Virus is presumed to becomplexed wither with complement or immunoglobulin and bound to eitherFc receptors or complement receptors or both. Thus, the virus exploitsthe normal mechanism of the immune system to retain antien memory forlong periods. During the course of the disease, active infection oflymphocytes occurs primarily at these sites. It has been calculated thatduring the asymptomatic phase of infection, the pool of virus in thiscompartment is more than 10 fold larger than that contained in T cellsand monocytes. (Cavert et al., 1997, “Kinetics of respnse in lymphoidtissues to antiretroviral therapy of HIV-1 infection”, Science276:960-4). In current HIV treatment modalities, multiple anti-viralagents are combined to reduce the viral load and to avoid escape ofresistant variants. A likely limitation of this therapy lies innon-compliance with the therapy and during such intervals, residualvirus is free to mutate alowing the development of resistant variantsand thus circumventing the therapy process. While the viral load in theFDC compartment is dramatically impacted during multiple drug therapy,the drugs themselves are largely directed at viral repliction machinery,and not at the non-replicating virus on FDC surfaces. Therefore, theviral reservoir on FDCs can serve as a re-inoculum following cessationof drug therapy. Moreover, the FDCs can convert neutralized virus to aninfectious form furhter underscoring the importance of these cells toHIV pathogenesis.

[0092] Since inhibition of the LT pathway can cause FDCs to releaseimmune complexes from the cell surface, HIV in the form of an immunecomplex could also be released. It would be desirable to release all ofthe HIV load in this compartment immediately prior to commencement ofmultiple therapy type regimes as the eleased virus should either beprocessed and removed from the body or upon infection, it would besensitive to the drug therapy. Such a combination could reduce theresidual viral load to very low levels possibly effecting a cure. Inthis case, either LTβ-R/Ig or blocking antibodies to either the ligandor the receptor would be useful. A poltential treatment proptocol wouldinvolve initiating drug therapy, and then within several days, releaseany bound virus with one or several treatmens with LT pathwayinhibitors. Once the viral load was reduced, further treatment with LTdirected agents would not be required.

[0093] While HIV is a particularly well studied example, it is likelythat other viruses reside or hide on FDC's in a quiescent state awaitingfor some event such as an immunolical disturbance which leads to largeamounts of additional antigen load, and consequently the release ofbound virus from FDCs and virus reemergence. Therefore, this inventionrelates to any means of Lt pathway inhibition to avoid the complicationsof FDC bound virus.

[0094] This discovery has significant implications for a number ofdiseases which rely on the presentation of antigen on dendritic cells,and response generated by memory B cells. LTα1/β2 signaling effectivelyand serves as an example of a therapeutically useful anti-LTα blockingmonoclonal. Additionally, an anti-human LT alpha directed monoclonalantibody entitled AOD12 was able to block LTα1/β2 signaling well yet incontrast to most anti-human LT alpha monoclonal antibodies, it waspoorly effective against LTα alone. These monoclonal antibodies wereobtained following immunization of mice with soluble LTα1/β2 ligandleading to the discovery of monoclonal antibodies with uniquespecificity. Furthermore, we maintain that anti-LTα monoclonalantibodies with specificity directed preferentially against the LT≢1/β2complex will be found only following this form of immunization and notvia immunization with LT alpha alone and hence comprises a unique classof anti-LTα antibodies.

EXAMPLES

[0095] Materials and Methods

[0096] Mice

[0097] Timed pregnant Balb/c mice were purchased from Jackson Laboratory(Bar Harbor, ME), housed under conventional barrier protection, andhandled in accordance with institutional guidelines. Receptor-Igproteins or mAbs were injected into the tail vein (iv) of pregnant mice.Progeny of these mice and 5 week old female Balb/c mice (purchased fromJackson Laboratory, Bar Harbor, ME) were injected with fusion proteinsvia the intraperitoneal (ip) route.

[0098] Fusion Proteins and Antibodies

[0099] Fusion proteins comprised of the extracellular domain of eithermurine LTβ-R, human TNF-R55 or human LFA-3 (which does not bind murineCD2) fused to the hinge, C_(H)2 and C_(H)3 domains of human IgG1 wereprepared as described (Force et al., J. Immunol., 155, pp. 5280-5288(1995); Miller et al., J. Exp. Med., 178, pp. 211-222 (1993)). Purifiedhuman IgG1 used as a control was purchased from Protos Immunoresearch(San Francisco, Calif.). MR1, anti-mouse CD40 ligand antibody, waspurchased from Pharmingen (San Diego, Calif.).

[0100] Antibodies (MOMA-1, ED3) specific for markers expressed by mousemetallophilic macrophages (MM), (ED3 recognizes sialoadhesin), orspecific for mouse reticular fibroblasts (ER-TR-7) were purchased fromSerotec (Oxon, UK). Antibodies specific for mouse B220, CD4, andMadCAM-1 (MECA 367) were purchased from Pharmingen (San Diego, Calif.).An antibody (ER-TR-9) specific for a marker expressed by mouse marginalzone macrophages was provided by Dr. Reina Mebius (Vrije Universiteit,Amsterdam). Antibodies (FDC-M1 and FDC-M2) specific for mouse folliculardendritic cell (FDC) have been described previously (Maeda et al., J.Immunol., 148, pp. 2340-2347 (1992)). Anti-mouse CR1 antibody (whichalso stains FDC) was kindly supplied by Dr. Randolph J. Noelle(Dartmouth Medical School). Detection of peripheral lymph node addressin(PNAd) utilized the antibody MECA 79 (cell culture supernatant derivedfrom cells purchased from ATCC, Rockville, Md.).

[0101] Antigens and Immunizations

[0102] Mice were immunized ip with 100 μl of a 10% suspension of SRBC(purchased from the Colorado Serum Company). This is equivalent to1-5×10⁸ SRBC per immunization.

[0103] Immunohistochemistry

[0104] Spleen and lymph nodes were frozen in OCT embedding medium(Miles, Elkhart, IN) and mounted for cryostat sectioning. Sections 7-10mm thick were dried and fixed with acetone. Sections were incubated withconjugated antibodies for 1 hr at room temperature in a humidified boxafter dilution in Tris buffered saline buffer A (TBS-A, 0.05M Tris,0.15M NaCl, 0.05% Tween-20 (v/v), 0.25% bovine serum albumin (BSA)),rinsed in TBS-B (0.05M Tris, 0.15M NaCl, 0.05% Tween-20) and fixed 1 minin methanol before initiating the enzymatic reaction. Horseradishperoxidase (HRP) and alkaline phosphatase (AP) activities were developedusing the DAB tablet substrate kit (Sigma, St. Louis, Mo.) and5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT,Sigma), respectively. Tissue sections were fixed for 5 min in methanoland counter stained with Giemsa (Fluka, Buchs, Switzerland).

[0105] Fluorescence Image Analysis

[0106] For immunofluorescence staining, frozen sections were acetonefixed, air dried and preblocked with 5 μg/ml anti-CD16/CD32 Fc block(Pharmingen, San Diego Calif.) in Tris Buffered Saline with 0.25% BSA,0.05% Tween-20 and 10% heat aggregated rabbit serum. Sections werestained in the same buffer using the following mAbs and detectionreagents: 10 μg/ml biotinylated anti-B220 mAb (Pharmingen) followed by20 μg/ml streptavidin-FITC (Southern Biotechnology Associates,Birmingham, Ala.); 10 μg/ml of MECA 367 followed by 10 μg/ml PE-goatF(ab′)₂ anti-rat IgG (Southern Biotechnology Associates); culturesupernatant of MECA79 followed by 20 μg/ml FITC-mouse anti-rat IgM(Pharmingen); 20 μg/ml anti-Sialoahesin mAb followed by 10 μg/ml PE-goatF(ab′)₂ anti-rat IgG (Southern Biotechnology Associates), 50 μg/mlbiotinylated PNA (Vector Laboratories, Burlingame, Calif.) followed by10 μg/ml streptadvidin-PE (Southern Biotechnology Associates); 1:5dilution of mAb MOMA-1 cell culture supernatant followed by 20 μg/mlFITC-mouse anti-rat IgM (Pharmingen). Some sections were stained withmultiple mAbs simultaneously to allow image overlay analysis. Allsections were viewed under 50× optics and photographed using EktachromeP1600 (Kodak, Rochester, NY) or captured as separate red and green imagefiles as described (Rennert et al, J. Exp. Med. (November 1996, inpress)).

[0107] Hemagglutination Assays

[0108] Serial dilutions of sera were made in 96 well microtiter plates(Costar, Cambridge, Mass.) in PBS/1% glucose. The SRBC-specific IgMtiter was determined by adding 25 l of a 10% SRBC suspension in eachwell and incubating the plate 1 hr in a humidified 37° C. incubator. ForSRBC-specific IgG, sera was incubated for 30 min at 37° C. with 20μl/well of 1% 2-mercaptoethanol (vol/vol) (Bio-Rad, Richmond, Calif.) toeliminate IgM pentamers. Then 25 μl/well of a 10% SRBC suspension wasadded, followed by 25 μl/well of a 10 mg/ml solution (in PBS/1% glucose)of goat anti-mouse IgG (Southern Biotechnology, Birmingham, Ala.) as acrosslinker agent for hemagglutination. The titer was determined as thereciprocal of last serum dilution for which hemagglutination is clearlyapparent.

[0109] ELISAs

[0110] Analyses for receptor-Ig in plasma used mAbs specific for murineLTβ-R (Browning et al, manuscript in preparation), LFA-3 (Miller, etal., J. Exp. Med., 178, pp. 211-222 (1993)) or the CH3 domain of humanIgG₁ (CDG5, prepared at Biogen) directly immobilized (10 μg/ml) on 96well microtiter plates for capture, and donkey anti-human IgG₁-HorseRadish Peroxidase (HRP) for detection (Jackson ImmunoResearch, WestGrove Pa. 1:4000 dilution).

[0111] Production of Soluble LTβ-R Molecules

[0112] The LTβ-R blocking agents in one embodiment of this inventioncomprise soluble LTβ receptor molecules. FIG. 1 shows the sequence ofthe extracellular portion of the human LTβ-R, which encodes the ligandbinding domain. Using the sequence information in FIG. 1 and recombinantDNA techniques well known in the art, functional fragments encoding theLTβ-R ligand binding domain can be cloned into a vector and expressed inan appropriate host to produce a soluble LTβ-R molecule. Soluble LTβ-Rmolecules that can compete with native LTβ receptors for LT ligandbinding according to the assays described in applicants copending U.S.application Ser. No. 08/505,606, filed Jul. 21, 1995, are selected asLTβ-R blocking agents.

[0113] A soluble LTβ receptor comprising amino acid sequences selectedfrom those shown in FIG. 1 may be attached to one or more heterologousprotein domains (“fusion domain”) to increase the in vivo stability ofthe receptor fusion protein, or to modulate its biological activity orlocalization.

[0114] Preferably, stable plasma proteins—which typically have ahalf-life greater than 20 hours in the circulation—are used to constructthe receptor fusion proteins. Such plasma proteins include but are notlimited to: immunoglobulins, serum albumin, lipoproteins,apolipoproteins and transferrin. Sequences that can target the solubleLTβ-R molecule to a particular cell or tissue type may also be attachedto the LTβ-R ligand binding domain to create a specifically-localizedsoluble LTβ-R fusion protein.

[0115] All or a functional portion of the LTβ-R extracellular region(FIG. 1) comprising the LTβ-R ligand binding domain may be fused to animmunoglobulin constant region like the Fc domain of a human IgG1 heavychain (Browning et al., J. Immunol., 154, pp. 33-46 (1995)). Solublereceptor-IgG fusion proteins are preferable, and are commonimmunological reagents, and methods for their construction are known inthe art (see e.g., U.S. Pat. No. 5,225,538 incorporated herein byreference).

[0116] A functional LTβ-R ligand binding domain may be fused to animmunoglobulin (Ig) Fc domain derived from an immunoglobulin class orsubclass other than IgG1. The Fc domains of antibodies belonging todifferent Ig classes or subclasses can activate diverse secondaryeffector functions. Activation occurs when the Fc domain is bound by acognate Fc receptor. Secondary effector functions include the ability toactivate the complement system, to cross the placenta, and to bindvarious microbial proteins. The properties of the different classes andsubclasses of immunoglobulins are described in Roitt et al., Immunology,p. 4.8 (Mosby-Year Book Europe Ltd., 3d ed. 1993).

[0117] Activation of the complement system initiates cascades ofenzymatic reactions that mediate inflammation. The products of thecomplement system have a variety of functions, including binding ofbacteria, endocytosis, phagocytosis, cytotoxicity, free radicalproduction and solubilization of immune complexes.

[0118] The complement enzyme cascade can be activated by the Fc domainsof antigen-bound IgG1, IgG3 and IgM antibodies. The Fc domain of IgG2appears to be less effective, and the Fc domains of IgG4, IgA, IgD andIgE are ineffective at activating complement. Thus one can select a Fcdomain based on whether its associated secondary effector functions aredesirable for the particular immune response or disease being treatedwith the LTβ-R-Ig fusion protein.

[0119] If it would be advantageous to harm or kill the LT ligand-bearingtarget cell, one could select an especially active Fc domain (IgG1) tomake the LTβR-Ig fusion protein. Alternatively, if it would be desirableto target the LTβR-Fc fusion to a cell without triggering the complementsystem, an inactive IgG4 Fc domain could be selected.

[0120] Mutations in Fc domains that reduce or eliminate binding to Fcreceptors and complement activation have been described (S. Morrison,Annu. Rev. Immunol., 10, pp. 239-65 (1992)). These or other mutationscan be used, alone or in combination, to optimize the activity of the Fcdomain used to construct the LTβ-R-Ig fusion protein.

[0121] The production of a soluble human LTβ-R fusion protein comprisingligand binding sequences fused to a human immunoglobulin Fc domain(hLTβ-R-Ig) is described in Example 1. One CHO line made according toExample 1 that secretes hLTβ-R-Fc is called “hLTβ-R;hGl CHO#14”. Asample of this line was deposited on Jul. 21, 1995 with the AmericanType Culture Collection (ATCC) (Rockville, Md.) according to theprovisions of the Budapest Treaty and was assigned the ATCC accessionnumber CRL11965.

[0122] The production of a soluble murine LTβ-R fusion molecule(LTβ-R-Ig) is described in Example 2. A CHO line made according toExample 2 that secretes LTβ-R-Ig is called “mLTβ;R-hGl CHO#1.3.BB”. Asample of this line was deposited on Jul. 21, 1995 with the AmericanType Culture Collection (ATCC) (Rockville, Md.) according to theprovisions of the Budapest Treaty and was assigned the ATCC accessionnumber CRL11964.

[0123] All restrictions on the availability to the public of the aboveATCC deposits will be irrevocably removed upon the granting of a patenton this application.

[0124] Different amino acid residues forming the junction point of thereceptor-Ig fusion protein may alter the structure, stability andultimate biological activity of the soluble LTβ receptor fusion protein.One or more amino acids may be added to the C-terminus of the selectedLTβ-R fragment to modify the junction point with the selected fusiondomain.

[0125] The N-terminus of the LTβ-R fusion protein may also be varied bychanging the position at which the selected LTβ-R DNA fragment iscleaved at its 5′ end for insertion into the recombinant expressionvector. The stability and activity of each LTβ-R fusion protein may betested and optimized using routine experimentation and the assays forselecting LTβ-R blocking agents described herein.

[0126] Using the LTβ-R ligand binding domain sequences within theextracellular domain shown in FIG. 1, amino acid sequence variants mayalso be constructed to modify the affinity of the soluble LTβ receptoror fusion protein for LT ligand. The soluble LTβ-R molecules of thisinvention can compete for surface LT ligand binding with endogenous cellsurface LTβ receptors. It is envisioned that any soluble moleculecomprising a LTβ-R ligand binding domain that can compete with cellsurface LTβ receptors for LT ligand binding is a LTβ-R blocking agentthat falls within the scope of the present invention.

[0127] Source of Anti-LTβ-R Antibodies

[0128] In another embodiment of this invention, antibodies directedagainst the human LTβ receptor (anti-LTβ-R Abs) function as LTβ-Rblocking agents. The anti-LTβ-R Abs of this invention can be polyclonalor monoclonal (mAbs) and can be modified to optimize their ability toblock LTβ-R signalling, their in vivo bioavailability, stability, orother desired traits.

[0129] Polyclonal antibody sera directed against the human LTβ receptorare prepared using conventional techniques by injecting animals such asgoats, rabbits, rats, hamsters or mice subcutaneously with a human LTβreceptor-Ig fusion protein (Example 1) in complete Freund's adjuvant,followed by booster intraperitoneal or subcutaneous injection inincomplete Freund's. Polyclonal antisera containing the desiredantibodies directed against the LTβ receptor are screened byconventional immunological procedures.

[0130] Mouse monoclonal antibodies (mAbs) directed against a human LTβreceptor-Ig fusion protein are prepared as described in applicantscopending U.S. application Ser. No. 08/505,606, filed Jul. 21, 1995. Ahybridoma cell line (BD.A8.AB9) which produces the mouse anti-humanLTβ-R mAb BDA8 was deposited on Jan. 12, 1995 with the American TypeCulture Collection (ATCC) (Rockville, Md.) according to the provisionsof the Budapest Treaty, and was assigned the ATCC accession numberHB11798. All restrictions on the availability to the public of the aboveATCC deposits will be irrevocably removed upon the granting of a patenton this application.

[0131] Various forms of anti-LTβ-R antibodies can also be made usingstandard recombinant DNA techniques (Winter and Milstein, Nature, 349,pp. 293-99 (1991)). For example, “chimeric” antibodies can beconstructed in which the antigen binding domain from an animal antibodyis linked to a human constant domain (e.g. Cabilly et al., U.S. Pat. No.4,816,567; Morrison et al., Proc. Natl. Acad. Sci. U.S.A., 81, pp.6851-55 (1984)). Chimeric antibodies reduce the observed immunogenicresponses elicited by animal antibodies when used in human clinicaltreatments.

[0132] In addition, recombinant “humanized antibodies” which recognizethe LTβ-R can be synthesized. Humanized antibodies are chimerascomprising mostly human IgG sequences into which the regions responsiblefor specific antigen-binding have been inserted (e.g. WO 94/04679).Animals are immunized with the desired antigen, the correspondingantibodies are isolated, and the portion of the variable regionsequences responsible for specific antigen binding are removed. Theanimal-derived antigen binding regions are then cloned into theappropriate position of human antibody genes in which the antigenbinding regions have been deleted. Humanized antibodies minimize the useof heterologous (inter-species) sequences in human antibodies, and areless likely to elicit immune responses in the treated subject.

[0133] Construction of different classes of recombinant anti-LTβ-Rantibodies can also be accomplished by making chimeric or humanizedantibodies comprising the anti-LTβ-R variable domains and human constantdomains (CH1, CH2, CH3) isolated from different classes ofimmunoglobulins. For example, anti-LTβ-R IgM antibodies with increasedantigen binding site valencies can be recombinantly produced by cloningthe antigen binding site into vectors carrying the human μ chainconstant regions (Arulanandam et al., J. Exp. Med., 177, pp. 1439-50(1993); Lane et al., Eur. J. Immunol., 22, pp. 2573-78 (1993);Traunecker et al., Nature, 339, pp. 68-70 (1989)).

[0134] In addition, standard recombinant DNA techniques can be used toalter the binding affinities of recombinant antibodies with theirantigens by altering amino acid residues in the vicinity of the antigenbinding sites. The antigen binding affinity of a humanized antibody canbe increased by mutagenesis based on molecular modeling (Queen et al.,Proc. Natl. Acad. Sci. U.S.A., 86, pp. 10029-33 (1989); WO 94/04679).

[0135] It may be desirable to increase or to decrease the affinity ofanti-LTβ-R Abs for the LTβ-R depending on the targeted tissue type orthe particular treatment schedule envisioned. For example, it may beadvantageous to treat a patient with constant levels of anti-LTβ-R Abswith reduced ability to signal through the LT-β pathway forsemi-prophylactic treatments. Likewise, inhibitory anti-LTβ-R Abs withincreased affinity for the LTβ-R may be advantageous for short-termtreatments.

[0136] Source of Anti-Surface LT Ligand Antibodies

[0137] Another preferred embodiment of this invention involvescompositions and methods which comprise antibodies directed against LTligand that function as LTβ-R blocking agents. As described above forthe anti-LTβ-R Abs, anti-LT ligand antibodies that function as LTβ-Rblocking agents can be polyclonal or monoclonal, and can be modifiedaccording to routine procedures to modulate their antigen bindingproperties and their immunogenicity.

[0138] The anti-LT antibodies of this invention can be raised againsteither one of the two LT subunits individually, including soluble,mutant, altered and chimeric forms of the LT subunit. If LT subunits areused as the antigen, preferably they are LTβ subunits. If LTα subunitsare used, it is preferred that the resulting anti-LTα antibodies bind tosurface LT ligand and do not cross-react with secreted LTα or modulateTNF-R activity (according to the assays described in applicantscopending U.S. application Ser. No. 08/505,606, filed Jul. 21, 1995).

[0139] Alternatively, antibodies directed against a homomeric (LTβ) or aheteromeric (LTα/β) complex comprising one or more LT subunits can beraised and screened for activity as LTβ-R blocking agents. Preferably,LTα1/β2 complexes are used as the antigen. As discussed above, it ispreferred that the resulting anti-LTα1/β2 antibodies bind to surface LTligand without binding to secreted LTα and without affecting TNF-Ractivity.

[0140] The production of polyclonal anti-human LTα antibodies isdescribed in applicants' co-pending application (WO 94/13808).Monoclonal anti-LTα and anti-LTβ antibodies have also been described(Browning et al., J. Immunol., 154, pp. 33-46 (1995)).

[0141] Mouse anti-human LTβ mAbs were prepared as described inapplicants copending U.S. application Ser. No. 08/505,606, filed Jul.21, 1995. A hybridoma cell line (B9.C9.1) which produces the mouseanti-human LTβ-R mAb B9 was deposited on Jul. 21, 1995 with the AmericanType Culture Collection (ATCC) (Rockville, Md.) according to theprovisions of the Budapest Treaty, and was assigned the ATCC accessionnumber HB11962.

[0142] Monoclonal hamster anti-mouse LTα/β antibodies were prepared asdescribed in applicants copending U.S. application Ser. No. 08/505,606,filed Jul. 21, 1995. A hybridoma cell line (BB.F6.1) which produces thehamster anti-mouse LTα/β mAb BB.F6 was deposited on Jul. 21, 1995 withthe American Type Culture Collection (ATCC) (Rockville, Md.) accordingto the provisions of the Budapest Treaty, and was assigned the ATCCaccession number HB11963.

[0143] All restrictions on the availability to the public of the aboveATCC deposits will be irrevocably removed upon the granting of a patenton this application.

[0144] Use of Soluble LTβ-R-Ig to Inhibit the Immunological Functions ofthe Surface LT Complex.

[0145] We now show effects of a surface LT binding reagent, a fusionprotein comprised of the extracellular domain of murine LTβ-R and thehinge, CH2 and CH3 domains of human IgG1 (LTP-R-Ig), on the generationand character of immunoglobulin responses, on the maintenance of thecellular organization of secondary lymphoid tissues including effects onthe differentiation state of follicular dendritic cells and germinalcenter formation, and on addressin expression levels which influencecell trafficking.

[0146] Multiple Injections of Mice with LTβ-R-Ig Alters the Organizationof Splenic Lymphocytes and the Expression of Functional Markers bySplenic Marginal Zone Cells.

[0147] The effect of surface LT blockade on the structure of the spleenwas examined by giving mice six consecutive weekly injections ofLTβ-R-Ig. Mice were then immunized with SRBC and given an additionalinjection of LTβ-R-Ig 4 days later. Mice were sacrificed on day 10 afterSRBC injection. Immunohistochemical staining of frozen spleen sectionsrevealed several histologic changes. The follicles which comprise thesplenic B cell compartment in normal mice are no longer discretefollowing LTβ-R-Ig treatment. Instead B cells are now organized in adiffuse band surrounding the T cell areas (FIG. 2B), and the boundarybetween the T and B cell zones is disrupted (FIG. 2B). In contrast, inthe control LFA-3-Ig treated mice the splenic B cell follicles arediscrete and there is a clear demarcation between T and B cell areas(FIG. 2A).

[0148] Expression of cell surface markers recognized by the monoclonalantibodies ER-TR-9 and MOMA-1 is absent from two distinct macrophagepopulations residing in the splenic marginal zone of LTβ-R-Ig treatedmice. ER-TR-9 is known to stain a marker on MZM (Dijkstra et al.,Immunol., 55, 23-30 (1985)) and MOMA-1 stains a marker on metallophilicmacrophages (Kraal and Janse, Immunol., 58, 665-669, (1986)) (FIGS.2D,F, respectively). These markers are expressed on cells in control(LFA-3-Ig) treated mice (FIGS. 2C,E). Expression of sialoadhesin,another marker of MOMA-1+ macrophages in the murine splenic marginalzone, is also absent in LTβ-R-Ig treated mice (data not shown).

[0149] The antibody MECA-367 binds the adhesion molecule and mucosaladdressin MAdCAM-1, originally described on endothelial cells in Peyer'spatches, mesenteric lymph nodes, the intestinal mucosa and laminapropria (Briskin et al., Nature, 363, pp. 461-464 (1993); Nakache etal., Nature, 337, pp. 179-181 (1989)). MAdCAM-1 expression has also beendescribed in the splenic marginal zone (presumably expressed on theendothelial cells of the small terminal arterioles opening onto themarginal sinus) and on the reticular meshwork within the germinalcenters (Kraal et al., Am. J. Pathol., 147, pp. 763-771 (1995)) (FIG.2G). MECA 367 staining of sections from LTβ-R-Ig treated mice show thatMAdCAM-1 expression has been extinguished in the spleen (FIG. 2H).

[0150] Likewise, the staining by the ER-TR-7 antibody (Van Vliet et al.,Cytochem., 34, pp. 883-890 (1986)) which delineates a population ofreticular fibroblasts in the marginal zone (FIG. 2I), is abnormallydistributed and stronger in the white pulp of the LTβ-R-Ig than LFA-3-Igtreated animals (FIG. 2J). The changes observed in LTβ-R-Ig treated micewere independent of antigen exposure as the pattern of staining wasidentical in LTβ-R-Ig treated unimmunized mice (data not shown).

[0151] Germinal Center Formation is Ablated and Follicular DendriticCells are not Detected in the Spleens of LTβ-R-Ig Treated Mice.

[0152] To determine at the histologic level whether multiple injectionsof mice of LTβ-R-Ig affects the immune response to SRBC, an analysis ofgerminal center (GC) formation and follicular dendritic cell (FDC)distribution in response to antigen priming was performed. Frozen spleensections from mice pretreated multiple times with LTβ-R-Ig or LFA-3-Igas described for FIG. 2, were stained with peanut agglutinin (PNA) todelineate the GCs and with the FDC-M1 antibody to detect FDC, a cellularcomponent required for GC formation (Schriever and Nadler, Adv.Immunol., 51, pp. 243-284 (1992); Tew et al., Immunol. Rev., 117, pp.185-211 (1990)). The CD40-CD40 ligand interaction has also been shown tobe critical for GC formation (Foy et al., J. Exp. Med., 180, pp. 157-163(1994)). Thus for comparison, a group of mice was treated with MR1, ananti-mouse CD40 ligand antibody, following an injection protocol whichhas previously been shown to inhibit GC formation (Han et al., J.Immunol., 155, pp. 556-567 (1995)). Ten days after SRBC challenge, micetreated with the control LFA-3-Ig protein developed numerous PNA brightGC in the spleen (FIG. 3A). GC were not detected in the spleen ofLTβ-R-Ig or MR1-treated mice (FIGS. 3B,C respectively). However, theeffect of MR1 and LTβ-R-Ig can be distinguished by two additionalobservations. The staining for FDCs (FDC-M1) within the GC (FIG. 3D) isabsent in the spleen of LTβ-R-Ig treated mice (FIG. 3E) but stillpresent in the spleen of MR1-treated mice (FIG. 3F). Similarobservations were made using the FDC-M2 antibody to stain FDCs (data notshown). Thus LTβ-R-Ig treatment results in the phenotypic alteration ofFDC in the spleen and the failure to form GC.

[0153] In addition to staining GC, PNA also stains the marginal zone inthe spleen of normal mice. Such staining was also noted in LFA-3-Igtreated (FIG. 3A) and MR1-treated mice (FIG. 3C), but was absent in thespleen of LTβ-R-Ig treated mice (FIG. 3B).

[0154] The expression of sialoadhesin, MOMA-1, ER-TR-9, ER-TR-7 andMAdCAM-1 in the spleen of MR1-treated mice was also shown to be normal(data not shown), further distinguishing the molecular effects ofinterfering with CD40 and LTP-R signaling.

[0155] Kinetics of LTβ-R-Ig Induced Alterations of Splenic LymphocyteOrganization and Marginal Zone Cell Markers Expression.

[0156] The number of LTβ-R-Ig injections required to affect lymphocyteorganization and expression of marginal zone cell markers in the spleenwas analyzed. Mice were injected ip with LTβ-R-Ig either once ormultiple times as indicated in Table 1. Some mice were then alsoimmunized with SRBC on the day of the last LTβ-R-Ig injection. B220 andCD4 expression on B and T cells, respectively, and staining with PNA(for GC) and MECA367 (for MAdCAM-1), MOMA-1, ER-TR-9, and FDC-Ml wasassessed on frozen spleen sections from treated mice. The kinetics ofdisappearance for the staining of metallophilic macrophages, marginalzone macrophages, MAdCAM-1, GCs and FDCs are shown to be distinct.

[0157] One injection of LTβ-R-Ig is sufficient to eliminate MAdCAM-1staining a week later. Following three weekly LTP-R-Ig injections,staining for GCs and FDCs is not detected and the T/B lymphocytecompartments are disrupted. A minimum of TABLE 1 Effect of LTβ-R-Ig onsplenic organization and germinal center formation in response to SRBCin adult mice. Number of LTβ-R-Ig Metallophilic Marginal zone MAdCAM-1Germinal Injections T/B cell organization macrophages* macrophages*expression* Centers§ FDC* 0  normal +++ +++ +++ +++ +++ 1  normal ++ +++− + + 2  slightly abnormal + +++ − + +/− 3‡ disrupted + ++ − ND ND 4‡disrupted +/− ++ − ND ND 5‡ disrupted − + − ND ND 6  disrupted − +/− − −− # mouse anti-rat Ig-peroxidase, respectively. Spleen sections werealso stained with the following anti-mouse antibodies: rat anti-marginalzone macrophages (ER-TR9), rat anti-metallophilic macrophages (MOMA-1),rat anti-MAdCAM-1 (MECA 367) and rat anti-FCD (FDC-M1), followed by amouse anti-rat Ig-peroxidase. An additional set of frozen sections wasstained with biotinylated peanut agglutinine (PNA-biotin), followed bystaining with streptavidin-peroxidase to detect germinal centers. #Observations were made on sections from at least 3 animals per group.

[0158] four LTβ-R-Ig injections are required to abolish the staining formetallophilic macrophages. Six LTβ-R-Ig injections do not completelyablate staining of marginal zone macrophages with ER-TR-9 antibody (alsoillustrated in FIG. 2D).

[0159] A more precise analysis of the rapid inhibition of MOMA-1,MAdCAM-1, FDC-M1, FDC-M2 and CR1 staining following a single injectionof LTβ-R-Ig was done in the absence of antigen (Table 2). TABLE 2Precise timing of LTβ-R-Ig effects on the MOMA-1, MAdCAM-1, CR1, FDC-M1and FDC-M2 staining DAYS AFTER A SINGLE INJECTION OF LTβ-R-Ig 0 1 3 5 710 14 MOMA-1* +++ +++ +++ +++ ++ ++ + MAdCAM-1* +++ +/− − − − − − CR1*+++ +++ ++ ++ ++ ++ + FDC-M1* +++ +/− − − − − − FDC-M2* +++ +/− − − − −−

[0160] Balb/c mice, 5-6 weeks received one ip injection of 100 μg ofLTβ-R-Ig or human Ig. Mice from each group were sacrificed on day 0, 1,3, 5, 7, 10 and 14. Frozen spleen sections were stained with thefollowing antibodies: rat anti-mouse metallophilic macrophages (MOMA-1),rat anti-mouse MAdCAM-1 (MECA 367), rat anti-mouse FDC (FDC-M1), ratanti-mouse FDC (FDC-M2) and biotin-labeled rat anti-mouse CR1 followedby a mouse anti-rat Ig-peroxidase (MOMA-1, MAdCAM-1, FDC-M1 and FDC-M2)or peroxidase-labeled streptavidin (CR1).

[0161] * The intensity of the staining was estimated by eye: normalstaining: +++, reduced staining ++, weak staining +, and no staining −.The staining intensity on sections from untreated animals and animalstreated with human Ig was taken as a reference for the normal staining.Sections from at least 2 animals per group were analyzed.

[0162] Balb/c mice which received a single ip injection of LTβ-R-Ig weresacrificed every day for fourteen days after injection and their spleenswere removed and frozen. Frozen spleen sections were stained withMOMA-1, anti-MAdCAM-1 (MECA-367), and FDC specific reagents: FDC-M1,FDC-M2 and anti-CR1 antibodies. One day after LTβ-R-Ig injection,staining with anti-MAdCAM-1 FDC-M1 and FDC-M2 reagents was greatlyreduced (Table 2). The faster inhibition of FDC-M1 staining in thisexperiment compared to results described in Table 1 may be due to theintensity of FDC-M1 staining which is stronger in immunized animals.Staining for CR1 was still detectable at day 14 indicating that the FDCwere still present on day 3 after treatment with LTβ-R-Ig but that theexpression of markers detected with FDC-M1 and FDC-M2 was extinguished.Thus LTPR-Ig treatment altered the FDC phenotype. Finally, MOMA-1staining was reduced but still detected at day 14.

[0163] Multiple LTβ-R-Ig treatments inhibit addressin expression in LN.

[0164] We examined addressin expression in LN of the progeny of timedpregnant Balb/c mice which were injected iv on days 14 and 17 ofgestation with 200 μg of receptor-Ig proteins. After birth the progenywere either untreated or injected once per week with 100 μg of LTβ-R-Ig,TNF-R55-Ig, or LFA-3-Ig ip. Fusion protein levels remained at or above10 μg/ml throughout life as determined by ELISA (data not shown).Immuno-histochemical staining with MECA367 and MECA79 showed thatMAdCAM-1 and peripheral LN addressins were entirely absent in mesentericLN from mice treated throughout life with LTβ-R-Ig (FIGS. 4A, B). SacralLN from these mice also lacked expression of all addressins and thecervical and iliac LN did not show peripheral lymph node (PNAd) staining(data not shown). The downregulation of addressin expression wasreversible, since expression recovered to normal levels in animals whichwere treated only in utero (FIGS. 4G, H). In mice treated throughoutlife with 100 ag/week TNF-R55-Ig or LFA-3-Ig, addressin expression in LNremained comparable to untreated mice (FIGS. 4C,D,E,F).

[0165] B Lymphocyte Positioning and Macrophage Marker Expression isAltered in LN of LTβ-R-Ig Treated Mice.

[0166] Antibodies which bind markers on macrophage populations in the LNsubcapsular sinus (analogous to the splenic marginal zone) were used todo immunohistochemical analysis of LN taken from mice which had beentreated during gestation and continuously after birth as described forFIG. 4 above with LTβ-R-Ig, TNF-R55-Ig, or LFA-3-Ig. Fluorescent imageswere analyzed using image analysis software. Sialoadhesin expression isshown to be diminished in LN of mice treated with soluble LTβ-R-Ig (FIG.5B), but not in LN of TNF-R55-Ig or LFA-3-Ig mice (FIGS. 5E, H). MOMA-1expression on macrophages in the subcapsular sinus was still detected inLN of mice treated with LTβ-R-Ig (FIG. 5C).

[0167] Effects of continuous LTβ-R-Ig treatment on lymphocyteorganization in LN were also evaluated. LN sections were stained withmAbs specific for the B cell marker B220 and the T cell marker CD4.Image analysis was used in order to identify areas of overlap of T and Bcell zones. Treatment with LTβ-R-Ig caused the dissolution of B cellfollicles such that the B cells were present in a diffuse band on theouter margin of the T cell area (FIG. 5A). Despite the dissolution oftheir follicular structure, B cells were not present within T cell areasof the LN, instead they appeared in areas not normally occupied bylymphocytes. A very similar pattern of T and B cell staining wasobserved in mice treated throughout life with 100 μg/week TNF-R55-Ig,but not LFA-3-Ig (FIG. 5D). Again B cell follicles were disrupted and Bcells were present in areas of the LN not usually found to containlymphocytes. Overlap of B cells with T cells was not observed.

[0168] LTβ-R-Ig Treatment of Mice Inhibits the IgM and IgG AntibodyResponse.

[0169] The failure of splenic GC to form following SRBC priming of micetreated multiple times with LTβ-R-Ig (as in FIG. 3) suggestedalterations in the humoral immune response of these mice. To test thisdirectly adult mice received six injections, once weekly, of LTβ-R-Ig orLFA-3-Ig and were then primed with SRBC. Mice were bled on days 7 and 14postimmunization and the presence of SRBC-specific IgM and IgG in thesera was analyzed using hemagglutination assays. Seven days after SRBCimmunization the IgM titer is normal but the IgG response is greatlydiminished in LTβ-R-Ig treated mice as compared to mice treated withhuman Ig or PBS. (FIG. 6A). On day 14 postimmunization, SRBC specificIgG still is not detected in the sera from LTβ-R-Ig treated mice and thetiter of SRBC specific IgM in these mice is also decreased by more thanhalf compared to human Ig or PBS treated mice (FIG. 6A).

[0170] Ten days after SRBC priming GCs are detected in the spleens ofmice treated once or twice with LTβ-R-Ig, however the number of GCs isgreatly diminished compared to controls (Table 1). When mice receivedtwo injections of LTβ-R-Ig, the first injection a week before SRBCpriming, and the second injection the same day as SRBC injection, theinhibition of the IgM and IgG response to SRBC at day 7 and day 14 (FIG.6B) is similar to that detected when mice received multiple LTβ-R-Iginjections (FIG. 6A). At day 30 postimmunization, SRBC-specific IgG isnot detected and the IgM levels are reduced by more than 80% compared tocontrols (FIG. 6B). Thus these LTβ-R-Ig treatment protocols resulted inthe complete inhibition of IgG responses, and an abbreviated/diminishedIgM response relative to the controls.

[0171] When mice received a single injection of LTβ-R-Ig on the same dayas SRBC priming, the level of the IgG and IgM responses to SRBC on day 7was comparable to that of the control groups (FIG. 6C). However, on day24 postimmunization the IgM and IgG titers are both reduced by 30%. Atday 34 after SRBC priming, the titer of SRBC-specific IgM is reduced by50% compared to control groups and SRBC-specific IgG could not bedetected (FIG. 6C). These data show that this LTβ-R-Ig treatmentprotocol resulted in the marked abbreviation/reduction of the levels ofboth an ongoing IgM and IgG response, that is that LTβ-R-Ig treatmentcan inhibit a humoral response which has already been initiated.

[0172] Antibody Mediated Diseases

[0173] Many organ-specific and multisystem autoimmune conditions involvepathological antibody responses. Such conditions include: MyastheniaGravis, autoimmune hemolytic anemia, Chagas' disease, Grave's disease,idiopathic thrombocytopenia purpura (ITP) Systemic Lupus Erythematosus(SLE), Wegener's Granulomatosis, Poly-arteritis Nodosa and RapidlyProgressive Crescentic Glomerulonephritis. (From Benjamini, et al.Immunology, A Short Course, (Wiley-Liss, New York 3d ed. (1996))Although the etiology of SLE is undefined, a fair amount is known aboutthe immunologic mechanism responsible for the pathology observed. Forunknown reasons, patients with SLE produce antibodies against nuclearcomponents of the body (antinuclear antibodies (ANA) notably againstnative double stranded DNA. Clinically the presence of these antibodiescorrelates best with the pathology of renal involvement in SLE. Theseantibodies complex with DNA apparently derived from the breakdown ofnormal tissue, and as in any immune—aggregate disease, such complexesform deposits trapped against the basement membrane of the glomeruli, inarteriolar walls and in joint synovial spaces. These complexes activatethe complement cascade and attract granulocytes. The subsequentinflammatory reaction is characterized as glomerulonephritis, withresulting damage to the kidneys leading to proteinuria and hematuria.

[0174] Lupus nephritis has been studied in murine models for decades.Recently, the therapeutic efficacy of a reagent specific for the murineCD40 ligand was evaluated in such a model (Mohan, et al., J. Immunol.,154, pp. 1470-1480 (1995)). The acceleration of lupus by the transfer ofcells which induce the production of pathogenic antibodies in vivo wasshown to be inhibited by administration of a monoclonal antibody whichblocks CD40/CD40 ligand interactions. Moreover a brief treatment oflupus mice with anti-CD40 ligand antibody had a sustained beneficialeffect on their spontaneous disease long after the antibody had beencleared from their systems. The experimentation indicated thatpathogenic B cells could not produce antibody even 9 months after thetherapy suggesting that there was a delay of the expansion of autoimmunememory B cells resulting in long-term therapeutic benefits. As we haveshown that reagents which block LTα/β/LTP-R interactions in vivo inhibitthe generation of antibody responses, alter the phenotype of FDC and theformation of germinal centers involved in optimal generation of B cellmemory, the LTα/α/LTβ-R blocking reagents of this invention will beuseful for treating or preventing SLE.

[0175] The normal immune response to some pathogenic infectious agentsalso elicits autoantibody responses that can become excessive andpresent a medical problem. One example is Chagas' disease, aninflammatory cardiomyopathy which develops in humans and experimentalanimals with chronic Trypanosoma cruzi infection. Among the possiblemechanisms involved in the pathogenesis of human Chagas'cardio-myopathy, induction of heart—specific autoimmune responses hasrecently received substantial experimental support. A recent study(Tibbetts, et al., J. Immunol., 152, pp. 1493-1499 (1994)) determinedthat cardiac antigen-specific antibodies are produced in T.Cruzi—infected C57B1/6 mice with heart disease. Upon infection with theBrazil strain of T. Cruzi, C57B1/6 mice develop a cardiomyopathy that ishistologically similar to that observed in chronically infected humans.Antisera from these mice react with three cardiac antigens while C57B1/6mice infected with the Guayas strain of T Cruzi which do not developcardiomyopathy did not produce such antibodies. These data indicate thatthese antibodies are specific markers of cardiomyopathy. Thus theability of LTβ-R blocking agents to inhibit damage mediated byautoantibodies can be assessed in such a rodent model.

[0176] Another example of cell destruction by autoantibodies generatedas a consequence of certain infectious diseases or for other unknownreasons is idiopathic thrombocytopenia purpura (ITP). In this conditionantibodies directed to platelets result in platelet destruction (bycomplement or phagocytic cells with Fc or C3b receptor) which may leadto bleeding. Therapeutics which will inhibit such antibody mediatedautoimmune reactions in vivo such as the LT-β-R blocking agents of thisinvention—which inhibit antibody generation—will be useful to treat orprevent these autoimmune diseases as well.

[0177] The normal immune response to some pathogenic infectious agentsalso elicits hypersensitivity reactions that can become excessive andpresent itself as a medical problem. The most prevalent example of typeI hypersensitivity is allergic reaction. These are mediated by IgEantibodies which bind via their Fc portion to receptors on mast cellsand basophils to trigger the release of pharmacologically active agentsthat mediate anaphylaxis. ITP and Goodpasture's syndrome are sometimesthought to be Type II reactions which occur when IgM or IgG antibodiesbind to antigen on the cell surface and activate the complement cascade.Granulocytes are then attracted to the site of activation, and damagefrom the release of lytic enzymes from their granules results in thedestruction of cells. Rheumatic arthritis is thought to result from atype III hypersensitivity reaction mediated by immune complexes ofantigen (in this case rheumatoid factor, an IgM autoantibody) that bindsto the Fc portion of normal IgG. These immune complexes participate incausing inflammation of joints and the damage characteristic of thisdisease. As these pathologies are mediated in part by antibodies,therapeutics which will inhibit the generation of antibody such as theLT-β-R blocking agents of this invention—will be useful for treating orpreventing these diseases as well.

[0178] Treatments Using LTβ-R Blocking Agents

[0179] The compositions of this invention will be administered at aneffective dose to treat the particular clinical condition addressed.Determination of a preferred pharmaceutical formulation and atherapeutically efficient dose regiment for a given application is wellwithin the skill of the art taking into consideration, for example, thecondition and weight of the patient, the extent of desired treatment andthe tolerance of the patient for the treatment.

[0180] Doses of about 1 mg/kg of a soluble LTβ-R are expected to besuitable starting points for optimizing treatment doses.

[0181] Determination of a therapeutically effective dose can also beassessed by performing in vitro experiments that measure theconcentration of the LTβ-R blocking agent required to coat target cells(LTβ-R or LT ligand-positive cells depending on the blocking agent) for1 to 14 days. The receptor-ligand binding assays described previously inapplicants copending U.S. application Ser. No. 08/505,606, filed Jul.21, 1995, can be used to monitor the cell coating reaction. LTβ-R or LTligand-positive cells can be separated from activated lymphocytepopulations using FACS. Based on the results of such in vitro bindingassays, a range of suitable LTβ-R blocking agent concentrations can beselected to test in animals.

[0182] Administration of the soluble LTβ-R molecules, anti-LT ligand andanti-LTβ-R Abs of this invention, alone or in combination, includingisolated and purified forms of the antibodies or complexes, their saltsor pharmaceutically acceptable derivatives thereof, may be accomplishedusing any of the conventionally accepted modes of administration ofagents which exhibit immunosuppressive activity.

[0183] The pharmaceutical compositions used in these therapies may alsobe in a variety of forms. These include, for example, solid, semi-solidand liquid dosage forms such as tablets, pills, powders, liquidsolutions or suspensions, suppositories, and injectable and infusiblesolutions. The preferred form depends on the intended mode ofadministration and therapeutic application. Modes of administration mayinclude oral, parenteral, subcutaneous, intravenous, intralesional ortopical administration.

[0184] The soluble LTβ-R molecules, anti-LT ligand and anti-LTβ-R Abs ofthis invention may, for example, be placed into sterile, isotonicformulations with or without cofactors which stimulate uptake orstability. The formulation is preferably liquid, or may be lyophilizedpowder. For example, the soluble LTβ-R molecules, anti-LT ligand andanti-LTβ-R Abs of this invention may be diluted with a formulationbuffer comprising 5.0 mg/ml citric acid monohydrate, 2.7 mg/ml trisodiumcitrate, 41 mg/ml mannitol, 1 mg/ml glycine and 1 mg/ml polysorbate 20.This solution can be lyophilized, stored under refrigeration andreconstituted prior to administration with sterile Water-For-Injection(USP).

[0185] The compositions also will preferably include conventionalpharmaceutically acceptable carriers well known in the art (see forexample Remington's Pharmaceutical Sciences, 16th Edition, 1980, MacPublishing Company). Such pharmaceutically acceptable carriers mayinclude other medicinal agents, carriers, genetic carriers, adjuvants,excipients, etc., such as human serum albumin or plasma preparations.The compositions are preferably in the form of a unit dose and willusually be administered one or more times a day.

[0186] The pharmaceutical compositions of this invention may also beadministered using microspheres, liposomes, other microparticulatedelivery systems or sustained release formulations placed in, near, orotherwise in communication with affected tissues or the bloodstream.Suitable examples of sustained release carriers include semipermeablepolymer matrices in the form of shaped articles such as suppositories ormicrocapsules. Implantable or microcapsular sustained release matricesinclude polylactides (U.S. Pat. No. 3,773,319; EP 58,481), copolymers ofL-glutamic acid and ethyl-L-glutamate (Sidman et al., Biopolymers, 22,pp. 547-56 (1985)); poly(2-hydroxyethyl-methacrylate) or ethylene vinylacetate (Langer et al., J. Biomed. Mater. Res., 15, pp. 167-277 (1981);Langer, Chem. Tech., 12, pp. 98-105 (1982)).

[0187] Liposomes containing soluble LTβ-R molecules, anti-LT ligand andanti-LTβ-R Abs of this invention, alone or in combination, can beprepared by well-known methods (See, e.g. DE 3,218,121; Epstein et al.,Proc. Natl. Acad. Sci. U.S.A., 82, pp. 3688-92 (1985); Hwang et al.,Proc. Natl. Acad. Sci. U.S.A., 77, pp. 4030-34 (1980); U.S. Pat. Nos.4,485,045 and 4,544,545). Ordinarily the liposomes are of the small(about 200-800 Angstroms) unilamellar type in which the lipid content isgreater than about 30 mol. % cholesterol. The proportion of cholesterolis selected to control the optimal rate of soluble LTβ-R molecule,anti-LT ligand and anti-LTβ-R Ab release.

[0188] The soluble LTβ-R molecules, anti-LT ligand and anti-LTβ-R Abs ofthis invention may also be attached to liposomes containing other LTβ-Rblocking agents, immunosuppressive agents or cytokines to modulate theLTβ-R blocking activity. Attachment of LTβ-R molecules, anti-LT ligandand anti-LTβ-R Abs to liposomes may be accomplished by any knowncross-linking agent such as heterobifunctional cross-linking agents thathave been widely used to couple toxins or chemotherapeutic agents toantibodies for targeted delivery. Conjugation to liposomes can also beaccomplished using the carbohydrate-directed cross-linking reagent4-(4-maleimidophenyl) butyric acid hydrazide (MPBH) (Duzgunes et al., J.Cell. Biochem. Abst. Suppl. 16E 77 (1992)).

[0189] Advantages of Therapeutic Compositions Comprising LTβ-R BlockingAgents

[0190] The LTβ-R blocking agents of this invention are capable ofselectively inhibiting immune effector mechanisms. The inhibition ofantibody mediated immunity is inhibited by multiple mechanisms includingthe regulation of GC formation by influencing FDC function. Bothantibody and cell mediated immunity are inhibited in part by regulatingthe expression of addressins and thus influencing lymphocytetrafficking. Thus LTβ-R blocking agents will be useful in treatingconditions that are exacerbated by the activities of antibodies, oraberrant expression of addressing. The ability to selectively inhibitsuch immune mediated responses will be useful for treating abnormalitiesincluding various autoimmune and chronic inflammatory conditions. Asdiscussed above, treatment of such pathologic immune mediated conditionsgenerally employs immunomodulatory and immunosuppressive agents whichhave pleiotropic effects on a wide variety of cell types andimmunological responses. These non-specific immunosuppressive agents aregenerally required in high and often cytotoxic doses that cause adverseside effects.

[0191] The ability of a reagent which inhibits antibody responses toameliorate a pathologic immunological response is supported in therecent study of mouse lupus nephritis. In the latter study,administration of an antibody that blocks the CD40/CD40L pathway wasshown inhibit the acceleration of lupus nephritis produced upon transferof cells which induce the production of pathogenic antibodies in vivo,and have a sustained beneficial effect on spontaneous disease long afterthe antibody had been cleared from the system. These data indicate thatthe LTβ-R blocking agents of this invention will be useful insuppressing cellular rejection of tissue grafts and organ transplants byinhibiting processes leading to the generation of antibody responses.

[0192] The LTβ-R blocking agents of the compositions and methods of thisinvention can be modified to obtain a desirable level of LTβ-Rsignalling depending on the condition, disorder or disease beingtreated. It is envisioned that the absolute level of LTβ-R signallingcan be fine-tuned by manipulating the concentration and the affinitiesof the LTβ-R blocking agents for their respective molecular targets.

[0193] For example, in one embodiment of this invention, compositionscomprising soluble LTβ-R molecules are administered to a subject. Thesoluble LTβ receptor can effectively compete with cell surface LTβreceptors for binding surface LT ligands. The ability to compete withsurface LT ligands depends on the relative concentrations of the solubleand the cell surface LTβ-R molecules, and on their relative affinitiesfor ligand binding.

[0194] Soluble LTβ-R molecules harboring mutations that increase ordecrease the binding affinity of that mutant soluble LTβ-R with surfaceLT ligand can be made using standard recombinant DNA techniques wellknown to those of skill in the art. Large numbers of molecules withsite-directed or random mutations can be tested for their ability to actas LTβ-R blocking agents using routine experimentation and thetechniques described herein.

[0195] Similarly, in another embodiment of this invention, antibodiesdirected against either the LTβ receptor or one or more of the LT ligandsubunits function as LTβ-R blocking agents. The ability for theseantibodies to block LTβ receptor signalling can be modified by mutation,chemical modification or by other methods that can vary the effectiveconcentration or activity of the antibody delivered to the subject.

[0196] The ability to diminish LTβ-R signalling without completelyinhibiting it may be important for establishing or maintaining reducedlevels of LTβ-R signalling that support normal immune function whileinhibiting antibody or cell mediated responses which are exaggerated orabnormal.

[0197] Disruption of the LTα gene in a mouse leads to aberrantperipheral lymphoid organ development (De Togni et al., Science, 264,pp. 703-707 (1994)). Such mice lacked lymph nodes and their spleenslacked the usually clear demarcation between T and B cell-rich regionsin the follicles. We believe that this phenotype is associated with lossof surface LT-induced LTβ-R signalling because similar phenotypes havenot been observed by modulating TNF-R activity. The ability toselectively or to partially block the LTβ-R pathway may thus be usefulin treating abnormal development of lymphoid-like structures resultingfrom chronic inflammation associated with mis- or over-expression ofsignalling by the LTβ-R pathway.

[0198] Antibodies are critical mediators of immune responses topathologic agents. Thus the absolute inhibition of antibody responsesmay not be desirable in certain circumstances. For example, antibodiesare required to mediate resistance to infections by extracellularbacteria such as pneumococci and hemophilus.

[0199] The ability to influence the level of antibody generated byblocking LTβ-R signalling may be important in maximizing the beneficialresults which can be achieved by treatments with the LTβ-R blockingagents of this invention.

[0200] The terapeutic methods of the invention involve selectivelyinhibiting responses that are dependent in whole or in part on the LT-βpathway. The particular therapeutic uses of the claimed invention dependupon the relevant etiological mechanism of either the process to beinhibited, or the medically desirable process to be promoted, as will beapparent to those of skill in the art. Thus, the methods of theinvention involve, in various embodiments, administering atherapeutically effective amount of a blocking agent of the LT-β-R, orLT-β. The protein used in these methods may be either full lengthproteins, fragments of the protein, or fusion fragments. In otherembodiments, the methods involve the administration of a solublefragment, such as a soluble lymphotoxin-β receptor. In other preferredembodiments, the claimed invention relates to the administration ofantibodies against the LT-β-R or LT-β. The blocking agents of theinvention may be administered concurrently with a therapeuticallyeffective amount of a second compound which exerts a medically desirableeffect.

[0201] For example, in certain methods for the treatment of AIDS and/orHIV, one may desire to co-administer additional antiviral agents knownin the art. For example, AZT, or protease inhibitors. Particularlypreferred may be the administration of blocking agents of the invention,more preferably, LT-β-R/IgG fusion protein, in combination with AIDS“cocktail” therapy. These drug “cocktails” involve the administration toa patient of multiple drugs to reduce the amount of virus in thepatient's systems.

[0202] Compositions of the invention may be formulated according tostandard practice, such as prepared in a carrier vehicle. The termpharmaceutically acceptable carrier refers to one or more organic orinorganic ingredients, natural or synthetic, which may facilitate theadministration of the blocking agents of the invention to a patient.Suitable carriers are known to those of orgdinarry skill in the art.

[0203] Any of the compositions of the invention may be administered inany manner which is medically acceptable. This may include injections,by parenteral routes, such as intravenous, intravascular, intraarterial,subcutaneious, intramuscular, intratumor, intraperitoneal,intraentriculare, intraepidural, or others, as well as oral, nasal,opthalmic, rectal or topical. sustained release administrateion is alsospecifically included in the invention, b means such as depot injectionsor implants. Localized delivery may also be diesirable. Modes ofadministration are easily defined by those skilled in the art.

[0204] The blocking agents of the LT pathway which are useful in theclaimed invention are intended to include functional derivatives of thesoluble LT-β-R, antibodies claimed herein. Functional derivativesinclude fragments, variants, analogs or chemical derivatives of amolecule. A fragment of a molecule, such as any of the antigens of thepresent invention is meant to reer to any polypeptide subset of themolecule. A vriant of such molecule is meant to refer to a naturallyoccurring molecule substantially similar to either the entire molecule,or a fragment thereof. An analog of the molecule refers to a non-naturalmolecule substantially similar to either the entire molecule or afragment thereof.

[0205] Variants of the blocking agents of the invention differ fromnaturally occurring agetns in amino acid sequence, or in ways that donot involve sequence, or both. Variants in amino acid sequence areproduced when one or more amino acids in the naturally occuringmolecules is substituted with a different natural amino acid, an aminoacid derivative, or a non-native amino acid. Paticularly preferredvariants include the naturally occuring proteins, or biologically activefragments of the naturally occuring proteins, whose sequencese differfrom the wild type sequence by one or more conservative amino acidsubstitutions. Such substitutions are well known by those skilled in theart, and typically have a minimal influence on the secondary structureand hydrophobic nature of the blocking agent.

[0206] In other embodiments, variants with amino acid substitutionswhich are less conservative may also result in desired derivatives,e.g., by causing changes in charge, conformation and other biologicalproperties. Such sustitutions would include for example, substitution ofhydrophilic residues for a hydrophobic residues, substitution of acysteine or a protline for another residue, substitution of a residuehaving a small side chain for a residue having a bulky side chain, orsubstitution of a residue having a net positive charge for a residuehaving a net negative charge. When the result of a given substitutioncannot be predicted with certainty, the derivatives may be readilyassayed according to the methods disclosed herein to determine thepresence or absence of the desired characteristics.

[0207] Variants within the scope of the invention include proteins andpeptides with amino acid sequences having at least eighty percenthomology with the blocking agents of the invention. More preferably thesequence homology is at least ninety percent, or at least ninety-fivepercent. For the purposes of determining homology the lenth ofcomparison sequences will generally be at least 8 amino acid residues,usaually at least 20 amino acid residues. Variants within the scope ofthe invention als include any blocking agent which 1) has an amino acidsequence which is at least forty percent homologous to the swequence oftrhe blocking agent, and aslso which, 2) after being placed in anoptimal alignment with the sequence of the blocking agent of theinvention, has at least 80% of its cystein residues aligned with thecysteins of the blocking agent of the invention.

[0208] Also within the invention are agents which specifically bind tothe blocking agents of the invention, including ligands and antibodies.

[0209] The following are examples which illustrate the soluble LTβreceptors, anti-LT ligand and anti-LTβ-R antibodies of this inventionand the methods used to characterize them. These examples should not beconstrued as limiting: the examples are included for purposes ofillustration and the present invention is limited only by the claims.

EXAMPLE 1 Preparation of Soluble Human LTβ Receptors as ImmunoglobulinFc Fusion Proteins

[0210] The sequence of a human cDNA clone isolated from a library ofhuman 12p transcribed sequences derived from a somatic cell hybrid(Baens et al., Genomics, 16, pp. 214-18 (1993)), was entered intoGenBank and was later identified as the sequence which encodes humanLTβ-R. The sequence of this full-length human LTβ-R cDNA clone has beenavailable since 1992 as GenBank entry L04270.

[0211] The extracellular domain of LTβ-R up to the transmembrane region(FIG. 1) was amplified by PCR from a cDNA clone using primers thatincorporated NotI and SalI restriction enzyme sites on the 5′ and 3′ends, respectively (Browning et al., J. Immunol., 154, pp. 33-46(1995)). The amplified product was cut with NotI and SalI, purified andligated into a NotI-linearized vector pMDR901 along with a SalI-NotIfragment encoding the Fc region of human IgG1. The resultant vectorcontained the dihydrofolate reductase gene and the LTBR-Ig fusionprotein driven by separate promoters.

[0212] The vector was electroporated into CHO dhfr cells andmethotrexate-resistant clones were isolated as per standard procedures.The LTβ-R-Ig was secreted into the medium and an ELISA assay was used toselect for cell lines producing the highest level of the receptor fusionprotein. A high-producing cell line was grown to large numbers and theconditioned medium collected. The pure LTβ receptor fusion protein wasisolated by Protein A Sepharose Fast Flow affinity chromatography(Pharmacia).

EXAMPLE 2 Preparation of Soluble Murine LTβ Receptors as ImmunoglobulinFusion Proteins

[0213] A complete cDNA clone of the murine LTβ-R was prepared byligating a 5′ NotI/ApaLI and 3′ ApaLI/NotI fragments from two partialcDNA isolates into the NotI site of pcDNA3 (InVitrogen, San Diego,Calif.). The sequence of this cDNA clone is accessible as GenBank entryU29173. No coding sequence differences were noted when compared withanother sequence entry for murine LTβ-R found in GenBank entry L38423.

[0214] A soluble murine LTβ-R/human IgG1 fusion protein was prepared byPCR amplification of the full length mLTβ-R cDNA clone as a template andthe primers 5′AACTGCAGCGGCCGCCATGCGCCTGCCC 3′ and5′GACTTTGTCGACCATTGCTCCTGGCTCTGGGGG 3′. The amplified product waspurified and cut with NotI and SalI and ligated with a SalI/NotI humanIgG1 Fc fragment into NotI-linearized and phosphatase-treated SAB132 toform JLB 122. For stable expression, the NotI cassette containing themurine LTβ-R-Ig fragment was transferred into the NotI site of pMDR901forming PSH001 and the vector was transfected into CHO cells asdescribed (Browning et al., J. Immunol., 154, pp. 33-46 (1995)). Cellclones secreting murine LTβ-R-Ig were identified by ELISA analysis. Thepurified receptor fusion protein was isolated from CHO cell supernatantsby Protein A Sepharose Fast Flow chromatography (Pharmacia) and isutilized in the examples which follow.

EXAMPLE 3 Immunohistochemical Analysis of Spleen Following MultipleInjections of Mice with LTβ-R-Ig

[0215] 4-5 week old mice received six injections, one per week, ofLTβ-R-Ig or LFA-3-Ig (100 μg ip), and were immunized with SRBC on theday of the sixth fusion protein injection. Mice then received anadditional injection of LTβ-R-Ig or LFA-3-Ig on day 4 after challengewith SRBC. The animals were sacrificed on day 10 after challenge withSRBC and organs were harvested for analysis of structure. The leftcolumn of FIG. 2 represents spleen sections from animals treated withLFA-3-Ig (A, C, E, G, I) and the right column from animals treated withLTβ-R-Ig (B, D, F, H, J). Acetone-fixed frozen spleen sections weredouble stained with biotinylated anti-mouse B220 labeled and anti-mouseCD4 antibodies (A and B), followed by a corresponding second stainingwith alkaline phosphatase-labeled streptavidin (purple blue, darkstaining) and horseradish peroxidase-labeled mouse anti-rat Ig with(light brown staining), respectively. Another set of frozen sectionswere stained with ER-TR-9 (to detect MZM, C and D), MOMA-1 (to detectmetallophilic macrophages, E and F), MECA-367 (specific for MAdCAM-1, Gand H), and ER-TR-7 (to stain reticular fibroblasts, I and J)antibodies, followed by a second staining with a horseradishperoxidase-labeled mouse anti-rat Ig (brown staining). These picturesare representative staining of sections from a minimum of six animals.Magnification 10×.

EXAMPLE 4 Effect of LTβ-R-Ig and Anti-CD40 Ligand on GC Formation andFDC Staining.

[0216] Animals were treated as described in example 3 with LTβ-R-Ig orLFA-3-Ig. Another group of animals was treated with MR1 (anti-mouse CD40ligand, 250 μg/injection, intraperiteonally) on day −1, day 1 and day 3,received SRBC on day 0 and were killed on day 10. Acetone-fixed spleensections of animals treated with LFA-3-Ig (FIG. 3 left column, A and D),or LTβ-R-Ig (middle column, B and E), or MR1 (right column, C and F)were stained with biotin-labeled peanut agglutinin (PNA, upper row, A, Band C) or with FDC-Ml (lower row, D, E and F), followed by a secondstaining with a horseradish peroxidase-labeled streptavidin andhorseradish peroxidase-labeled mouse anti-rat Ig, respectively (brownstaining). PNA staining of the marginal zone is indicated by an arrow inA and C. GC formation is indicated by a white star in A. Staining forFDC is indicated by a black arrow in D and F. These pictures arerepresentative staining of sections from at least four animals.Magnification 10×.

EXAMPLE 5

[0217] Addressin Expression in LN of Mice Treated in Utero andContinuously Post-Birth with LTβ-R-Ig

[0218] These experiments used the progeny of timed pregnant Balb/c micewhich were injected iv on days 14 and 17 of gestation with 200 μg ofreceptor-Ig proteins. After birth the progeny were injected ip once perweek with 100 ug of LTβ-R-Ig, TNF-R55-Ig, or LFA-3-Ig. Fusion proteinlevels remained at or above 10 μg/ml throughout life as determined byELISA (data not shown). FIG. 4: Panels A, B, G, H staining of lymphnodes from mice treated with LTβ-R-Ig. Panels C,D staining of lymphnodes from mice treated with LFA-3-Ig, Panels E,F staining of lymphnodes from mice treated with TNF—R55-Ig. Panels A,C,E,G are mesentericlymph nodes stained with the antibody MECA367 to detect the mucosaladdressing MAdCAM-1. Panels B,D,F,H are peripheral (brachial) lymphnodes stained with the antibody MECA79 specific for peripheral LNaddressins (PNAds). Panels G,H are lymph nodes from 6 week old miceexposed to LTβ-R-Ig in utero only. All images are 50×magnification.

EXAMPLE 6 Lymphocyte Positioning and Expression of Macrophage Markers inLN of Mice Treated in Utero and Continuously Post-Birth with LTβ-R-Ig.

[0219] Mice were treated in utero and continuously postbirth asdescribed for example 5 with LTβ-R-Ig, TNF-R55-Ig, or LFA-3-Ig. LNsections were then stained with antibodies specific for markersexpressed by macrophages or with mabs specific for the B cell markerB220 and the T cell marker CD4. Image analysis was used in order toidentify areas of overlap of T and B cell zones. FIG. 5: Panels A,D,Gare B220/CD4 staining of LN from LTβ-R-Ig, LFA-3-Ig and TNF-R55-Igtreated mice respectively. Fluorescent images were analyzed using imageanalysis software. Panels B,E,H are staining for sialoadhesin and panelsC,F,I are staining for MOMA-1.

EXAMPLE 7 Effect of LTβ-R-Ig Treatment on the Antibody Response to SRBC.

[0220] Balb/c mice were injected with either LTβ-R-Ig, human Ig or PBSas follows: FIG. 6A: mice received six injections as described for FIG.2, example 3. Animals were bled day 7 (black bars) and day 14 (stripedbars) after SRBC immunization. 6B: Animals received the fusion proteinson day −7 and day 0. SRBC were given on day 0 and the animals were bledon day 7 (black bars), day 14 (striped bars) and day 30 (white bars).6C: Animals received the fusion proteins once on day 0, at the same timeas the SRBC immunization. The blood was collected on day 7 (black bars),day 14 (striped bars) and day 34 (grey bars).

[0221] The titer of SRBC-specific IgM and IgG was determined byanalyzing the sera in hemagglutination assays. The titer is defined asthe reciprocal of the last serum dilution for which hemagglutination isdetected and is represented on a log base 2 scale (1=dilution {fraction(1/15)} of the sera). Results are represented as the mean of 4 differentanimals per group with standard deviations.

1 1 1 197 PRT Homo Sapien 1 Ser Gln Pro Gln Ala Val Pro Pro Tyr Ala SerGlu Asn Gln Thr Cys 1 5 10 15 Arg Asp Gln Glu Lys Glu Tyr Tyr Glu ProGln His Arg Ile Cys Cys 20 25 30 Ser Arg Cys Pro Pro Gly Thr Tyr Val SerAla Lys Cys Ser Arg Ile 35 40 45 Arg Asp Thr Val Cys Ala Thr Cys Ala GluAsn Ser Tyr Asn Glu His 50 55 60 Trp Asn Tyr Leu Thr Ile Cys Gln Leu CysArg Pro Cys Asp Pro Val 65 70 75 80 Met Gly Leu Glu Glu Ile Ala Pro CysThr Ser Lys Arg Lys Thr Gln 85 90 95 Cys Arg Cys Gln Pro Gly Met Phe CysAla Ala Trp Ala Leu Glu Cys 100 105 110 Thr His Cys Glu Leu Leu Ser AspCys Pro Pro Gly Thr Glu Ala Glu 115 120 125 Leu Lys Asp Glu Val Gly LysGly Asn Asn His Cys Val Pro Cys Lys 130 135 140 Ala Gly His Phe Gln AsnThr Ser Ser Pro Ser Ala Arg Cys Gln Pro 145 150 155 160 His Thr Arg CysGlu Asn Gln Gly Leu Val Glu Ala Ala Pro Gly Thr 165 170 175 Ala Gln SerAsp Thr Thr Cys Lys Asn Pro Leu Glu Pro Leu Pro Pro 180 185 190 Glu MetSer Gly Thr 195

What is claimed is:
 1. A method for altering the humoral immune responsein an animal comprising the step of a) administering a pharmaceuticalcomposition which comprises a therapeutically effective amount of aLT-β-R blocking agent.
 2. The method according to claim 1, wherein theLT-β-R blocking agent is selected from the group consisting of: solublelymphotoxin-β receptor, an antibody directed against LT-β receptor, andan antibody directed against surface LT ligand.
 3. The method accordingto claim 1, wherein the animal is a mammal.
 4. The method according toclaim 3, wherein the mammal is a human.
 5. The method according to claim2, wherein the LT-β-R blocking agent comprises a soluble lymphotoxin-βreceptor having a ligand binding domain that can selectively bind to asurface LT ligand.
 6. The method according to claim 5, wherein thesoluble lymphotoxin-β receptor comprises a human immunoglobulin Fcdomain.
 7. The method according to claim 2, wherein the LT-β-R blockingagent comprises a monoclonal antibody directed against LT-β receptor. 8.The method according to claim 7, wherein the composition is administeredin an amount sufficient to coat LT-β receptor-positive cells for about 1to about 14 days.
 9. The method according to claim 7, wherein the LT-β-Rblocking agent comprises anti-human LT-β-R mAb BDA8.
 10. The methodaccording to claim 2, wherein the LT-β-R blocking agent comprises amonoclonal antibody directed against surface LT ligand.
 11. The methodaccording to claim 10, wherein the composition is administered in anamount sufficient to coat surface LT ligand-positive cells for 1 to 14days.
 12. The method according to claim 10, wherein the antibody isdirected against a subunit of the LT ligand.
 13. The method according toclaim 12, wherein the LT-β-R blocking agent comprises anti-human LT-βmAb B9.
 14. The method according to claim 10, wherein the LT-β-Rblocking agent comprises a monoclonal antibody directed against a murinesurface LT ligand.
 15. The method of claim 1 further comprising apharmaceutically acceptable carrier or adjuvant.
 16. The methodaccording to claim 1, wherein the humoral immune response is inhibited.17. A pharmaceutical composition comprising a therapeutically effectiveamount of a LT-β-R blocking agent and a pharmaceutically acceptablecarrier.
 18. The composition according to claim 38, wherein the LT-β-Rblocking agent is selected from the group consisting of a solublelymphotoxin-β receptor, an antibody directed against LT-β receptor, andan antibody directed against surface LT ligand.
 19. A method forinhibiting LT-β-R signaling without inhibiting TNF-R signalingcomprising the step of administering to a subject an effective amount ofa LT-β-R blocking agent.
 20. The method according to claim 19, whereinthe LT-β-R blocking agent is selected from the group consisting of asoluble lymphotoxin-β receptor, an antibody directed against LT-βreceptor, and an antibody directed against surface LT ligand.
 21. Themethod according to claim 19, wherein the subject comprises one or morecells from a mammal.
 22. The method according to claim 21, wherein themammal is a human.
 23. The method according to claim 19, wherein theLT-β-R blocking agent comprises a soluble lymphotoxin-β receptor havinga ligand binding domain that can selectively bind to a surface LTligand.
 24. The method according to claim 23, wherein the solublelymphotoxin-β receptor further comprises a human immunoglobulin Fcdomain.
 25. The method according to claim 19, wherein the LT-β-Rblocking agent comprises a monoclonal antibody directed against LT-βreceptor.
 26. The method according to claim 22, wherein the LT-β-Rblocking agent comprises anti-human LT-β-R mAb BDA8.
 27. The methodaccording to claim 19, wherein the LT-β-R blocking agent comprises amonoclonal antibody directed against surface LT ligand.
 28. A method foraltering the association of immune complexes and B cell follicles in apatient comprising administering an amount of an LT-β-R blocking agentto said patient.
 29. The method of claim 28 wherein said patient isinfected with human immunodeficiency virus.
 30. The method of claim 28wherein said blocking agent is selected from the group consisting ofsoluble LT-β-R, an antibody directed against LT-β-R, and an antibodydirected against surface LT ligand.
 31. The method of claim 30 whereinsaid soluble LT-β-R has a ligand binding domain that can selectivelybind to a surface LT ligand.
 32. The method of claim 31 wherein saidsoluble receptor comprises a human immunoglobulin Fc domain.
 33. Themethod of claim 28 wherein the LT-β-R comprises a monoclonal antibodydirected against LT-β-R.
 34. The method of claim 33 wherein saidantibody is anit-human LT-β-R mAb BDA8.
 35. The method of claim 28further comprising a pharmaceutically acceptable carrier or adjuvant.36. A method of treating, preventing, or eliminating humanimmunodeficiency virus in a mammal comprising the step of administeringa pharmaceutical composition comprising a therapeutically effectiveamount of a LT-β-R blocking agent, and a pharmaceutically effectivecarrier.
 37. The method of claim 36 wherein the LT-β-R blocking agent isselected from the group consisting of soluble lymphotoxin-β-R, andantibody directed against LT-β-R, and an antibody directed againstsurface LT ligand.
 38. The method of claim 37 wherein the blocking agnetcomprises a soluble lymphotoxin-B-R comprising a ligand binding domainthat can selectively bind to a surface LT ligand.
 39. The method ofclaim 38 wherein the soluble receptor comprises a human immunoglobulinFc domain.
 40. The method of claim 36 wherein the LT-β-R blocking agentcomprises a monoclonal antibody directed against LT-β-R.
 41. The methodof claim 40 wherein the blocking agent comprises anti-human LT-β-R mAbBDA8.
 42. The method of claim 36 wherein the blocking agent comprises amonoclonal antibody directed against surface LT ligand.
 43. The methodof claim 36 further comprising the co-administration of an additionalanti-viral agent.
 44. The method of claim 28 wherein the B cells arefollicular dendritic cells.
 45. A pharmaceutical composition fortreating graft rejection comprising a therapeutically effective amountof a blocking agent of LT-β-R and a therapeutically effective amount ofa blocking agent of CD40L.
 46. The composition of claim 45 wherein theLT-β-R blocking agent is LT-β-R/IgG and the blocking agent of CD40L isan anti CD40L compound.
 47. A pharmaceutical composition for thetreatment of AIDS or HIV, comprising AZT, a protease inhibitor, and ablocking agent of LT-β-R.
 48. The composition of claim 47 wherein theblocking agent is LT-β-R/IgG fusion.
 49. The composition of claim 46wherein the anti-CD40L compound is a monoclonal antibody.
 50. Thecomposition of claim 49 wherein the antibody is 5c8.